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• W2107881559 abstract "Serotonin (5-HT) and leptin play important roles in the modulation of energy balance. Here we investigated mechanisms by which leptin might interact with CNS 5-HT pathways to influence appetite. Although some leptin receptor (LepRb) neurons lie close to 5-HT neurons in the dorsal raphe (DR), 5-HT neurons do not express LepRb. Indeed, while leptin hyperpolarizes some non-5-HT DR neurons, leptin does not alter the activity of DR 5-HT neurons. Furthermore, 5-HT depletion does not impair the anorectic effects of leptin. The serotonin transporter-cre allele (Sertcre) is expressed in 5-HT (and developmentally in some non-5-HT) neurons. While Sertcre promotes LepRb excision in a few LepRb neurons in the hypothalamus, it is not active in DR LepRb neurons, and neuron-specific Sertcre-mediated LepRb inactivation in mice does not alter body weight or adiposity. Thus, leptin does not directly influence 5-HT neurons and does not meaningfully modulate important appetite-related determinants via 5-HT neuron function. Serotonin (5-HT) and leptin play important roles in the modulation of energy balance. Here we investigated mechanisms by which leptin might interact with CNS 5-HT pathways to influence appetite. Although some leptin receptor (LepRb) neurons lie close to 5-HT neurons in the dorsal raphe (DR), 5-HT neurons do not express LepRb. Indeed, while leptin hyperpolarizes some non-5-HT DR neurons, leptin does not alter the activity of DR 5-HT neurons. Furthermore, 5-HT depletion does not impair the anorectic effects of leptin. The serotonin transporter-cre allele (Sertcre) is expressed in 5-HT (and developmentally in some non-5-HT) neurons. While Sertcre promotes LepRb excision in a few LepRb neurons in the hypothalamus, it is not active in DR LepRb neurons, and neuron-specific Sertcre-mediated LepRb inactivation in mice does not alter body weight or adiposity. Thus, leptin does not directly influence 5-HT neurons and does not meaningfully modulate important appetite-related determinants via 5-HT neuron function. 5-HT neurons do not express leptin receptors and are not directly leptin responsive 5-HT depletion does not impair anorectic leptin action Sertcre-mediated neuronal LepRb deletion does not affect DR leptin action, body weight, or adiposity Thus, leptin does not meaningfully influence appetite or body weight via brain 5-HT Leptin, a hormone secreted by adipocytes in proportion to fat mass, acts via the long form of the leptin receptor (LepRb) to regulate energy homeostasis (Myers et al., 2009Myers Jr., M.G. Munzberg H. Leinninger G.M. Leshan R.L. The geometry of leptin action in the brain: more complicated than a simple ARC.Cell Metab. 2009; 9: 117-123Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Neuronal-specific deletion of LepR promotes hyperphagia and obesity (Cohen et al., 2001Cohen P. Zhao C. Cai X. Montez J.M. Rohani S.C. Feinstein P. Mombaerts P. Friedman J.M. Selective deletion of leptin receptor in neurons leads to obesity.J. Clin. Invest. 2001; 108: 1113-1121Crossref PubMed Scopus (505) Google Scholar), and restoration of neuronal LepRb in LepR-deficient mice normalizes food intake and body weight (de Luca et al., 2005de Luca C. Kowalski T.J. Zhang Y. Elmquist J.K. Lee C. Kilimann M.W. Ludwig T. Liu S.M. Chua Jr., S.C. Complete rescue of obesity, diabetes, and infertility in db/db mice by neuron-specific LEPR-B transgenes.J. Clin. Invest. 2005; 115: 3484-3493Crossref PubMed Scopus (295) Google Scholar), indicating that leptin's effects on energy balance are achieved via brain LepRb. Within the brain, multiple populations of LepRb-expressing neurons that contribute to energy homeostasis have been identified, including (1) neurons of the hypothalamic arcuate nucleus (ARC) that express pro-opiomelanocortin (POMC) or agouti-related peptide (AgRP) (Balthasar et al., 2004Balthasar N. Coppari R. McMinn J. Liu S.M. Lee C.E. Tang V. Kenny C.D. McGovern R.A. Chua Jr., S.C. Elmquist J.K. et al.Leptin receptor signaling in POMC neurons is required for normal body weight homeostasis.Neuron. 2004; 42: 983-991Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar, van de Wall et al., 2008van de Wall E. Leshan R. Xu A.W. Balthasar N. Coppari R. Liu S.M. Jo Y.H. MacKenzie R.G. Allison D.B. Dun N.J. et al.Collective and individual functions of leptin receptor modulated neurons controlling metabolism and ingestion.Endocrinology. 2008; 149: 1773-1785Crossref PubMed Scopus (260) Google Scholar), (2) SF1-expressing neurons of the ventromedial nucleus of the hypothalamus (Dhillon et al., 2006Dhillon H. Zigman J.M. Ye C. Lee C.E. McGovern R.A. Tang V. Kenny C.D. Christiansen L.M. White R.D. Edelstein E.A. et al.Leptin directly activates SF1 neurons in the VMH, and this action by leptin is required for normal body-weight homeostasis.Neuron. 2006; 49: 191-203Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar), (3) dopaminergic neurons in the ventral tegmental area (Fulton et al., 2006Fulton S. Pissios P. Manchon R.P. Stiles L. Frank L. Pothos E.N. Maratos-Flier E. Flier J.S. Leptin regulation of the mesoaccumbens dopamine pathway.Neuron. 2006; 51: 811-822Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar, Hommel et al., 2006Hommel J.D. Trinko R. Sears R.M. Georgescu D. Liu Z.W. Gao X.B. Thurmon J.J. Marinelli M. DiLeone R.J. Leptin receptor signaling in midbrain dopamine neurons regulates feeding.Neuron. 2006; 51: 801-810Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar), (4) GABAergic neurons of the lateral hypothalamic area (LHA) (Leinninger et al., 2009Leinninger G.M. Jo Y.H. Leshan R.L. Louis G.W. Yang H. Barrera J.G. Wilson H. Opland D.M. Faouzi M.A. Gong Y. et al.Leptin acts via leptin receptor-expressing lateral hypothalamic neurons to modulate the mesolimbic dopamine system and suppress feeding.Cell Metab. 2009; 10: 89-98Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar), and (5) neurons within the nucleus of the solitary tract (Hayes et al., 2009Hayes M.R. Skibicka K.P. Leichner T.M. Guarnieri D.J. DiLeone R.J. Bence K.K. Grill H.J. Endogenous leptin signaling in the caudal nucleus tractus solitarius and area postrema is required for energy balance regulation.Cell Metab. 2009; 11: 77-83Abstract Full Text Full Text PDF Scopus (177) Google Scholar), among others (Myers et al., 2009Myers Jr., M.G. Munzberg H. Leinninger G.M. Leshan R.L. The geometry of leptin action in the brain: more complicated than a simple ARC.Cell Metab. 2009; 9: 117-123Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, Patterson et al., 2011Patterson C.M. Leshan R.L. Jones J.C. Myers Jr., M.G. Molecular mapping of mouse brain regions innervated by leptin receptor-expressing cells.Brain Res. 2011; 1378: 18-28Crossref PubMed Scopus (107) Google Scholar, Scott et al., 2009Scott M.M. Lachey J.L. Sternson S.M. Lee C.E. Elias C.F. Friedman J.M. Elmquist J.K. Leptin targets in the mouse brain.J. Comp. Neurol. 2009; 514: 518-532Crossref PubMed Scopus (372) Google Scholar). Leptin also acts in concert with the brain serotonin (5-HT) pathways, although important discrepancies exist regarding the mechanisms underlying this interaction. A variety of data suggest that leptin and 5-HT function independently but converge in the ARC, although recent publications report a role for direct leptin action via brain 5-HT neurons (Calapai et al., 1999Calapai G. Corica F. Corsonello A. Sautebin L. Di Rosa M. Campo G.M. Buemi M. Mauro V.N. Caputi A.P. Leptin increases serotonin turnover by inhibition of brain nitric oxide synthesis.J. Clin. Invest. 1999; 104: 975-982Crossref PubMed Scopus (160) Google Scholar, Harris et al., 1998Harris R.B. Zhou J. Redmann Jr., S.M. Smagin G.N. Smith S.R. Rodgers E. Zachwieja J.J. A leptin dose-response study in obese (ob/ob) and lean (+/?) mice.Endocrinology. 1998; 139: 8-19Crossref PubMed Scopus (0) Google Scholar, von Meyenburg et al., 2003von Meyenburg C. Langhans W. Hrupka B.J. Evidence for a role of the 5-HT2C receptor in central lipopolysaccharide-, interleukin-1 beta-, and leptin-induced anorexia.Pharmacol. Biochem. Behav. 2003; 74: 1025-1031Crossref PubMed Scopus (60) Google Scholar, Yadav et al., 2009Yadav V.K. Oury F. Suda N. Liu Z.W. Gao X.B. Confavreux C. Klemenhagen K.C. Tanaka K.F. Gingrich J.A. Guo X.E. et al.A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure.Cell. 2009; 138: 976-989Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, Yadav et al., 2011Yadav V.K. Oury F. Tanaka K. Thomas T. Wang Y. Cremers S. Hen R. Krust A. Chambon P. Karsenty G. Leptin-dependent serotonin control of appetite: temporal specificity, transcriptional regulation, and therapeutic implications.J. Exp. Med. 2011; 208: 41-52Crossref PubMed Scopus (70) Google Scholar, Yamada et al., 2003Yamada J. Sugimoto Y. Hirose H. Kajiwara Y. Role of serotonergic mechanisms in leptin-induced suppression of milk intake in mice.Neurosci. Lett. 2003; 348: 195-197Crossref PubMed Scopus (16) Google Scholar). We thus generated and utilized several mouse lines to resolve these issues and clarify the potential role for 5-HT neurons in leptin action. Analysis using a LepRb riboprobe (Elmquist et al., 1998Elmquist J.K. Bjorbaek C. Ahima R.S. Flier J.S. Saper C.B. Distributions of leptin receptor mRNA isoforms in the rat brain.J. Comp. Neurol. 1998; 395: 535-547Crossref PubMed Scopus (888) Google Scholar) revealed that, with the exception of the dorsal raphe nucleus (DR), brain regions containing 5-HT do not express Leprb mRNA in the rat (see Figures S1A–S1F′ available online) or mouse (Figures S1G–S1L′). Similarly, utilizing LepRbEGFP mice (Patterson et al., 2011Patterson C.M. Leshan R.L. Jones J.C. Myers Jr., M.G. Molecular mapping of mouse brain regions innervated by leptin receptor-expressing cells.Brain Res. 2011; 1378: 18-28Crossref PubMed Scopus (107) Google Scholar), we observed no colocalization of LepRb with 5-HT in the DR (Figures 1A and 1A ′, and Figure S1). In contrast, most DR neurons containing calbindin (Calb), a marker of interneurons, expressed LepRb (Figures 1B and 1B′); and, as previously reported, a small population of DR dopamine (DA) neurons also expressed LepRb (Figures S1M and S1M′) (Scott et al., 2009Scott M.M. Lachey J.L. Sternson S.M. Lee C.E. Elias C.F. Friedman J.M. Elmquist J.K. Leptin targets in the mouse brain.J. Comp. Neurol. 2009; 514: 518-532Crossref PubMed Scopus (372) Google Scholar). We also assayed leptin-induced STAT3 phosphorylation (pSTAT3), which is directly mediated by activated LepRb, to examine direct leptin action in the midbrain. Leptin-induced pSTAT3 was absent from all 5-HT-containing sites, with the exception of the DR, in the rat (Figures S1P–S1V′) and mouse (Figures S1W–S1AC′). Within the DR, leptin-induced pSTAT3 was absent from all 5-HT neurons in the rat (Figures 1C and 1C′, and Figures S1P–S1V′) and mouse (Figures 1D and 1D′, and Figures S1W–S1AC′). In contrast, most DR Calb neurons in the rat (Figures 1E and 1E′) and mouse (Figures 1F and 1F′) contained leptin-induced pSTAT3, as did a subpopulation of DR DA neurons in the rat (Figures S1N and S1N′) and mouse (Figures S1O and S1O′). Thus, although some Calb- and DA-expressing DR neurons contain functional LepRb, CNS 5-HT neurons do not. We also examined the ability of leptin to influence the membrane potential of identified DR 5-HT neurons in acute slice preparations from wild-type mice. We recorded from 26 DR neurons in current-clamp mode, after which recorded cells were filled with biocytin and slices were subjected to the post hoc immunohistochemistry (IHC) identification of 5-HT. None (0/5) of the recorded 5-HT-containing neurons demonstrated detectable changes in membrane potential in response to leptin (Figures 2A–2C ). Consistent with previous results from DR neurons not characterized post hoc by IHC (Yadav et al., 2009Yadav V.K. Oury F. Suda N. Liu Z.W. Gao X.B. Confavreux C. Klemenhagen K.C. Tanaka K.F. Gingrich J.A. Guo X.E. et al.A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure.Cell. 2009; 138: 976-989Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar), leptin inhibited the firing of a subpopulation (29% [6/21]) of DR neurons (Figures 2D–2F, p < 0.01). None of these leptin-responsive cells were 5-HT positive (Figure 2F). Yadav et al. reported increased expression of DR tryptophan hydroxylase 2 (Tph2), which mediates 5-HT synthesis, in old, but not young, Lepob/ob mice (Yadav et al., 2009Yadav V.K. Oury F. Suda N. Liu Z.W. Gao X.B. Confavreux C. Klemenhagen K.C. Tanaka K.F. Gingrich J.A. Guo X.E. et al.A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure.Cell. 2009; 138: 976-989Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar), which suggests that Tph2 expression changes in Lepob/ob mice are related to age, rather than to direct leptin effects. Indeed, unlike Yadav et al., 2009Yadav V.K. Oury F. Suda N. Liu Z.W. Gao X.B. Confavreux C. Klemenhagen K.C. Tanaka K.F. Gingrich J.A. Guo X.E. et al.A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure.Cell. 2009; 138: 976-989Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, we were not able to detect an effect of leptin on DR Tph2 in wild-type mice, either by acute peripheral (0.5, 2.0, or 5.0 mg/kg, i.p.) or central (5 μg ICV) leptin treatment (Figures S2A and S2B). Similarly, we were unable to detect any changes in DR Tph2 in fasted rats receiving continuous leptin treatment (a paradigm in which leptin induces changes in other appetite-associated neuropeptides [Ahima et al., 1999Ahima R.S. Kelly J. Elmquist J.K. Flier J.S. Distinct physiologic and neuronal responses to decreased leptin and mild hyperleptinemia.Endocrinology. 1999; 140: 4923-4931Crossref PubMed Scopus (215) Google Scholar]) (Figure S2C). Our data suggest that leptin does not directly or significantly modulate DR Tph2 expression. While leptin does not directly control CNS 5-HT neurons, we investigated potential indirect interactions between 5-HT- and LepRb-expressing cells using a transgenic mouse line in which LepRb neurons throughout the brain express the transneuronal tracer wheat germ agglutinin (Figure S3). Our observations indicate that an unidentified population of LepRb neurons lie in synaptic contact with DR 5-HT neurons (Figure S3). In order to address the possibility of 5-HT participation in leptin action via such an indirect mechanism, we utilized PCPA to selectively deplete brain 5-HT, and by consequence its metabolite 5-hydroxyindoleacetic acid, by ∼80% (Figure 3A ). Noradrenaline (NA) and DA were not affected (Figures 3B and 3C). This profound depletion of 5-HT did not interfere with the ability of leptin (5 mg/kg, i.p.) to attenuate food intake (Figure 3D). Thus, 5-HT is not required for the anorectic action of leptin. These data are consistent with the notion that leptin does not mediate its effects on appetite via LepRb neurons synapsing on 5-HT cells, and also with previous reports. Specifically, leptin exhibits normal anorectic efficacy in mice null for 5-HT2CR (Nonogaki et al., 1998Nonogaki K. Strack A.M. Dallman M.F. Tecott L.H. Leptin-independent hyperphagia and type 2 diabetes in mice with a mutated serotonin 5-HT2C receptor gene.Nat. Med. 1998; 4: 1152-1156Crossref PubMed Scopus (419) Google Scholar), and mice null for both leptin and 5-HT2CR show greater hyperphagia than mice with either mutation alone (Wade et al., 2008Wade J.M. Juneja P. MacKay A.W. Graham J. Havel P.J. Tecott L.H. Goulding E.H. Synergistic impairment of glucose homeostasis in ob/ob mice lacking functional serotonin 2C receptors.Endocrinology. 2008; 149: 955-961Crossref PubMed Scopus (37) Google Scholar). Furthermore, 5-HT requires the melanocortin pathway to influence food intake, whereas leptin affects appetite through both melanocortin-dependent and -independent pathways (Balthasar et al., 2004Balthasar N. Coppari R. McMinn J. Liu S.M. Lee C.E. Tang V. Kenny C.D. McGovern R.A. Chua Jr., S.C. Elmquist J.K. et al.Leptin receptor signaling in POMC neurons is required for normal body weight homeostasis.Neuron. 2004; 42: 983-991Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar, Dhillon et al., 2006Dhillon H. Zigman J.M. Ye C. Lee C.E. McGovern R.A. Tang V. Kenny C.D. Christiansen L.M. White R.D. Edelstein E.A. et al.Leptin directly activates SF1 neurons in the VMH, and this action by leptin is required for normal body-weight homeostasis.Neuron. 2006; 49: 191-203Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar, Heisler et al., 2002Heisler L.K. Cowley M.A. Tecott L.H. Fan W. Low M.J. Smart J.L. Rubinstein M. Tatro J.B. Marcus J.N. Holstege H. et al.Activation of central melanocortin pathways by fenfluramine.Science. 2002; 297: 609-611Crossref PubMed Scopus (409) Google Scholar, Lam et al., 2008Lam D.D. Przydzial M.J. Ridley S.H. Yeo G.S. Rochford J.J. O'Rahilly S. Heisler L.K. Serotonin 5-HT2C receptor agonist promotes hypophagia via downstream activation of melanocortin 4 receptors.Endocrinology. 2008; 149: 1323-1328Crossref PubMed Scopus (211) Google Scholar, Marsh et al., 1999Marsh D.J. Hollopeter G. Huszar D. Laufer R. Yagaloff K.A. Fisher S.L. Burn P. Palmiter R.D. Response of melanocortin-4 receptor-deficient mice to anorectic and orexigenic peptides.Nat. Genet. 1999; 21: 119-122Crossref PubMed Scopus (466) Google Scholar). Thus, leptin's effects on appetite and body weight are unlikely to be substantially 5-HT dependent. A recent paper by Yadav et al. reported that LepRb in 5-HT neurons is crucial for most leptin action (Yadav et al., 2009Yadav V.K. Oury F. Suda N. Liu Z.W. Gao X.B. Confavreux C. Klemenhagen K.C. Tanaka K.F. Gingrich J.A. Guo X.E. et al.A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure.Cell. 2009; 138: 976-989Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). Clearly, this conclusion is incompatible with the lack of LepRb/5-HT coexpression, the lack of direct leptin effect on DR 5-HT neurons, and the irrelevance of brain 5-HT content to the anorexic action of leptin that we have demonstrated here. Methodological factors may have promoted nonspecific signal in the LepRb localization studies performed by Yadav et al., as their depiction of LepRb abundance in the median raphe (MnR) as well as the DR is not consistent with previous reports of LepRb distribution (Elmquist et al., 1998Elmquist J.K. Bjorbaek C. Ahima R.S. Flier J.S. Saper C.B. Distributions of leptin receptor mRNA isoforms in the rat brain.J. Comp. Neurol. 1998; 395: 535-547Crossref PubMed Scopus (888) Google Scholar, Patterson et al., 2011Patterson C.M. Leshan R.L. Jones J.C. Myers Jr., M.G. Molecular mapping of mouse brain regions innervated by leptin receptor-expressing cells.Brain Res. 2011; 1378: 18-28Crossref PubMed Scopus (107) Google Scholar, Scott et al., 2009Scott M.M. Lachey J.L. Sternson S.M. Lee C.E. Elias C.F. Friedman J.M. Elmquist J.K. Leptin targets in the mouse brain.J. Comp. Neurol. 2009; 514: 518-532Crossref PubMed Scopus (372) Google Scholar). Yadav et al. reported that mice lacking LepRb in 5-HT transporter-cre (Sertcre)-expressing neurons are hyperphagic and obese (Yadav et al., 2009Yadav V.K. Oury F. Suda N. Liu Z.W. Gao X.B. Confavreux C. Klemenhagen K.C. Tanaka K.F. Gingrich J.A. Guo X.E. et al.A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure.Cell. 2009; 138: 976-989Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). Sert is broadly expressed in both 5-HT and non-5-HT neurons during development (Lebrand et al., 1998Lebrand C. Cases O. Wehrle R. Blakely R.D. Edwards R.H. Gaspar P. Transient developmental expression of monoamine transporters in the rodent forebrain.J. Comp. Neurol. 1998; 401: 506-524Crossref PubMed Scopus (160) Google Scholar, Narboux-Neme et al., 2008Narboux-Neme N. Pavone L.M. Avallone L. Zhuang X. Gaspar P. Serotonin transporter transgenic (SERTcre) mouse line reveals developmental targets of serotonin specific reuptake inhibitors (SSRIs).Neuropharmacology. 2008; 55: 994-1005Crossref PubMed Scopus (113) Google Scholar). We therefore investigated the possibility that the Sertcre eliminated LepRb in crucial populations of non-5-HT neurons (Figure 4). We crossed the Sertcre allele utilized by Yadav et al. onto the conditional Leprfl background and the ROSA26-reporter alleles that express EGFP/EYFP following cre-dependent excision (Mao et al., 1999Mao X. Fujiwara Y. Orkin S.H. Improved reporter strain for monitoring Cre recombinase-mediated DNA excisions in mice.Proc. Natl. Acad. Sci. USA. 1999; 96: 5037-5042Crossref PubMed Scopus (275) Google Scholar, McMinn et al., 2005McMinn J.E. Liu S.M. Liu H. Dragatsis I. Dietrich P. Ludwig T. Boozer C.N. Chua Jr., S.C. Neuronal deletion of Lepr elicits diabesity in mice without affecting cold tolerance or fertility.Am. J. Physiol. Endocrinol. Metab. 2005; 289: E403-E411Crossref PubMed Scopus (64) Google Scholar, Narboux-Neme et al., 2008Narboux-Neme N. Pavone L.M. Avallone L. Zhuang X. Gaspar P. Serotonin transporter transgenic (SERTcre) mouse line reveals developmental targets of serotonin specific reuptake inhibitors (SSRIs).Neuropharmacology. 2008; 55: 994-1005Crossref PubMed Scopus (113) Google Scholar). Sertcre;ROSA26-reporter mice displayed Sertcre-mediated recombination in non-5-HT neurons within the DR and elsewhere in the brain (Figure S4). We examined the potential colocalization of Sertcre-induced EGFP with leptin-stimulated pSTAT3 in Sertcre;ROSA26-EGFP mice (Figures 4A–4C); this revealed no colocalization of pSTAT3 with EGFP in the DR, although a few Sertcre/LepRb neurons were observed in the LHA (Figure S4). Given the modest number of Sertcre/LepRb neurons revealed by this analysis, we were surprised by the strong reported phenotype of Sertcre;Leprfl/fl (LeprSert) mice (Yadav et al., 2009Yadav V.K. Oury F. Suda N. Liu Z.W. Gao X.B. Confavreux C. Klemenhagen K.C. Tanaka K.F. Gingrich J.A. Guo X.E. et al.A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure.Cell. 2009; 138: 976-989Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar), and therefore bred Sertcre;Leprfl/+ animals to independently derived Leprfl/fl animals to generate LeprSert animals and littermate Leprfl/fl controls for analysis. Examination of leptin-stimulated pSTAT3 in the DR of control (Figures 4D–4F) and LeprSert (Figures 4G–4I) animals revealed similar levels and patterns of leptin-induced pSTAT3-IR in LeprSert and control animals, consistent with the lack of Sertcre colocalization with LepRb in the DR. We also examined the body weight and adiposity phenotypes of LeprSert and littermate control animals (Figures 4J–4L). Body and fat pad weights did not differ between LeprSert and control animals, in contrast to the previously reported approximately 60% increase in body weight and 3- to 4-fold increase in fat pad weight/body weight at similar ages (Yadav et al., 2009Yadav V.K. Oury F. Suda N. Liu Z.W. Gao X.B. Confavreux C. Klemenhagen K.C. Tanaka K.F. Gingrich J.A. Guo X.E. et al.A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure.Cell. 2009; 138: 976-989Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). Vertebral bones from LeprSert mice were also similar to controls (Figure 4M). As the Leprfl allele is prone to germline excision in a number of cre strains (generating the null LeprΔ allele) (van de Wall et al., 2008van de Wall E. Leshan R. Xu A.W. Balthasar N. Coppari R. Liu S.M. Jo Y.H. MacKenzie R.G. Allison D.B. Dun N.J. et al.Collective and individual functions of leptin receptor modulated neurons controlling metabolism and ingestion.Endocrinology. 2008; 149: 1773-1785Crossref PubMed Scopus (260) Google Scholar, and our unpublished data), we thus examined the possibility of such events in the LeprSert and littermate control animals from Figure 4 (Figure 4N). While LeprΔ is not distinguished from Leprfl by the standard three-primer multiplex genotyping paradigm to detect Leprfl and Lepr (Figure 4N, bottom panel) (McMinn et al., 2005McMinn J.E. Liu S.M. Liu H. Dragatsis I. Dietrich P. Ludwig T. Boozer C.N. Chua Jr., S.C. Neuronal deletion of Lepr elicits diabesity in mice without affecting cold tolerance or fertility.Am. J. Physiol. Endocrinol. Metab. 2005; 289: E403-E411Crossref PubMed Scopus (64) Google Scholar), separation of the primer sets into two reactions discerns Leprfl from the germline deleted (LeprΔ) allele (Figure 4N, top and middle panels). Our analysis revealed the presence of one germline-deleted LeprΔ allele in all of the LeprSert mice that we examined (i.e., our LeprSert mice were uniformly Sertcre;Leprfl/Δ while controls were Leprfl/fl), suggesting that germline deletion of Leprfl occurs uniformly in gametes containing both Sertcre and Leprfl. Given our present findings and the dramatic phenotype of the LeprSert mice (closely resembling mice completely devoid of leptin action) reported by Yadav et al., we surmise that this tendency toward germline deletion combined with subsequent inbreeding yielded LeprSert mice of the Sertcre;LeprΔ/Δ genotype (globally Lepr deficient) in their hands. While the LeprSert animals studied by Yadav et al. did not demonstrate overt diabetes as is often observed in animals devoid of LepRb, genetic background effects can suppress hyperglycemia in such animals, and this may have been the case in the mixed genetic background animals studied (Moritani et al., 2006Moritani M. Togawa K. Yaguchi H. Fujita Y. Yamaguchi Y. Inoue H. Kamatani N. Itakura M. Identification of diabetes susceptibility loci in db mice by combined quantitative trait loci analysis and haplotype mapping.Genomics. 2006; 88: 719-730Crossref PubMed Scopus (7) Google Scholar, Yadav et al., 2009Yadav V.K. Oury F. Suda N. Liu Z.W. Gao X.B. Confavreux C. Klemenhagen K.C. Tanaka K.F. Gingrich J.A. Guo X.E. et al.A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure.Cell. 2009; 138: 976-989Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). With regards to the more recent study in which LepRb was deleted in adults by means of an inducible Tph2-cre (for which no recombination mapping data were provided) (Yadav et al., 2011Yadav V.K. Oury F. Tanaka K. Thomas T. Wang Y. Cremers S. Hen R. Krust A. Chambon P. Karsenty G. Leptin-dependent serotonin control of appetite: temporal specificity, transcriptional regulation, and therapeutic implications.J. Exp. Med. 2011; 208: 41-52Crossref PubMed Scopus (70) Google Scholar), we postulate ectopic excision by this allele, given the substantial data demonstrating the lack of LepRb expression in 5HT neurons and the failure of Sertcre-mediated LepRb deletion to alter body energy balance. Neuron-specific LepRb deletion data have revealed no single pathway purported to mediate the majority of appetitive leptin action, with the exception of the recently suggested 5-HT system (Yadav et al., 2009Yadav V.K. Oury F. Suda N. Liu Z.W. Gao X.B. Confavreux C. Klemenhagen K.C. Tanaka K.F. Gingrich J.A. Guo X.E. et al.A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure.Cell. 2009; 138: 976-989Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, Yadav et al., 2011Yadav V.K. Oury F. Tanaka K. Thomas T. Wang Y. Cremers S. Hen R. Krust A. Chambon P. Karsenty G. Leptin-dependent serotonin control of appetite: temporal specificity, transcriptional regulation, and therapeutic implications.J. Exp. Med. 2011; 208: 41-52Crossref PubMed Scopus (70) Google Scholar). Here we clarify that brain 5-HT neurons do not express LepRb and do not, therefore, directly respond to leptin. Furthermore, our data reveal that brain 5-HT is not required for the anorectic action of leptin, and that neuron-specific excision of LepRb by Sertcre does not disrupt DR leptin action or the ability of leptin to regulate energy homeostasis. We conclude that leptin does not substantially influence energy balance via brain 5-HT neurons, and that the crucial population(s) of LepRb neurons for the control of energy balance remains to be precisely identified. LepRbEGFP mice were generated and propagated as previously described (Leinninger et al., 2009Leinninger G.M. Jo Y.H. Leshan R.L. Louis G.W. Yang H. Barrera J.G. Wilson H. Opland D.M. Faouzi M.A. Gong Y. et al.Leptin acts via leptin receptor-expressing lateral hypothalamic neurons to modulate the mesolimbic dopamine system and suppress feeding.Cell Metab. 2009; 10: 89-98Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). For studies with wild-type mice and rats, adult male pathogen-free C57BL/6 mice (27–30 g) or Sprague-Dawley rats (250–300 g) were used (Taconic or Charles River). All animals were individually housed with water and chow pellets available ad libitum (unless otherwise stated) in a light- (12 hr on/12 hr off) and temperature-controlled (21.5°C–22.5°C) environment. Sertcre and Leprfl mice were the generous gifts of Xiaoxi Zhuang (University of Chicago) and Streamson Chua (Albert Einstein College of Medicine), respectively. ROSA26-EGFP and ROSA26-EYFP animals were from the Jackson Laboratory. All procedures performed in the USA were in accordance with National Institutes of Health (NIH) guidelines on animal care and use and with the approval of the UCUCA; those performed in the UK were approved by the UK Home Office. Sertcre mice were genotyped via qPCR, using primers and probes for Cre and Ngf, as described (Leinninger et al., 2009Leinninger G.M. Jo Y.H. Leshan R.L. Louis G.W. Yang H. Barrera J.G. Wilson H. Opland D.M. Faouzi M.A. Gong Y. et al.Leptin acts via leptin receptor-expressing lateral hypothalamic neurons to modulate the mesolimbic dopamine system and suppress feeding.Cell Metab. 2009; 10: 89-98Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Leprfl was genotyped by conventional PCR (mLepR-105, mLepR-65A for Leprfl and Lepr+) in addition to or in combination with mLepR-105 and mLepR-106 for LeprΔ (McMinn et al., 2005McMinn J.E. Liu S.M. Liu H. Dragatsis I. Dietrich P. Ludwig T. Boozer C.N. Chua Jr., S.C. Neuronal deletion of Lepr elicits diabesity in mice without affecting cold tolerance or fertility.Am. J. Physiol. Endocrinol. Metab. 2005; 289: E403-E411Crossref PubMed Scopus (64) Google Scholar). For pSTAT3 assessment in rats, femoral vein catheters were inserted as done previously (Elias et al., 1998Elias C.F. Lee C. Kelly J. Aschkenasi C. Ahima R.S. Couceyro P.R. Kuhar M.J. Saper C.B. Elmquist J.K. Leptin activates hypothalamic CART neurons projecting to the spinal cord.Neuron. 1998; 21: 1375-1385Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar). Five days later, rats were injected with saline or recombinant rat leptin (Sigma, 0.25 mg/kg or 1.0 mg/kg, i.v.) (n = 4 per dose). For pSTAT3 assessment in C57BL/6 or Sertcre; reporter mice, saline or recombinant murine leptin (Peprotech or a generous gift of Amylin Pharmaceuticals) was administered (n = 3/genotype). Animals were deeply anesthetized and perfused transcardially with 0.9% saline followed by 10% formalin (Sigma); brain tissue was collected as previously described (Elias et al., 1998Elias C.F. Lee C. Kelly J. Aschkenasi C. Ahima R.S. Couceyro P.R. Kuhar M.J. Saper C.B. Elmquist J.K. Leptin activates hypothalamic CART neurons projecting to the spinal cord.Neuron. 1998; 21: 1375-1385Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar, Heisler et al., 2002Heisler L.K. Cowley M.A. Tecott L.H. Fan W. Low M.J. Smart J.L. Rubinstein M. Tatro J.B. Marcus J.N. Holstege H. et al.Activation of central melanocortin pathways by fenfluramine.Science. 2002; 297: 609-611Crossref PubMed Scopus (409) Google Scholar, Leinninger et al., 2009Leinninger G.M. Jo Y.H. Leshan R.L. Louis G.W. Yang H. Barrera J.G. Wilson H. Opland D.M. Faouzi M.A. Gong Y. et al.Leptin acts via leptin receptor-expressing lateral hypothalamic neurons to modulate the mesolimbic dopamine system and suppress feeding.Cell Metab. 2009; 10: 89-98Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, Munzberg et al., 2007Munzberg H. Jobst E.E. Bates S.H. Jones J. Villanueva E. Leshan R. Bjornholm M. Elmquist J. Sleeman M. Cowley M.A. et al.Appropriate inhibition of orexigenic hypothalamic arcuate nucleus neurons independently of leptin receptor/STAT3 signaling.J. Neurosci. 2007; 27: 69-74Crossref PubMed Scopus (66) Google Scholar) (see the Supplemental Experimental Procedures for details). For dual-label IHC, primary antibodies for pSTAT3 (1:250, rabbit, Cell Signaling), GFP (1:1,000, rabbit, Molecular Probes or 1:000, chicken, Abcam), 5-HT (1:1,000, goat, Immunostar), Calb (1:500, mouse, Sigma), or TH (1:1,000, mouse, Chemicon) were followed by secondary antibodies (Molecular Probes and Jackson Immunoresearch): FITC-conjugated donkey anti-chicken (for GFP, 1:200), Alexa Fluor 594 conjugated to donkey anti-goat (for 5-HT, 1:500), or Alexa Fluor 488 conjugated to donkey anti-mouse or anti-rabbit (for Calb, TH, and GFP, 1:500). For the analysis of LeprSert and control mice, 8-week-old male LeprSert (n = 9) and control littermate (n = 5) mice were weighed. A subset of mice (LeprSert = 5, control = 4) were implanted with ICV cannulae as described (Leinninger et al., 2009Leinninger G.M. Jo Y.H. Leshan R.L. Louis G.W. Yang H. Barrera J.G. Wilson H. Opland D.M. Faouzi M.A. Gong Y. et al.Leptin acts via leptin receptor-expressing lateral hypothalamic neurons to modulate the mesolimbic dopamine system and suppress feeding.Cell Metab. 2009; 10: 89-98Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Coordinates to the lateral ventricle were, relative to Bregma, A/P −0.34, M/L −1.0, and D/V −2.4. For treatment, the dummy was replaced with an injector to deliver 3 μl of PBS or leptin (1 mg/mL; Amylin Pharmaceuticals) at a rate of 1 μl per minute. Mice were perfused 1 hr after treatment and brains were sectioned and analyzed for pSTAT3 and GFP as described above. Mice that were not cannulated were also perfused at 9 weeks of age. Gonadal fat was dissected and weighed postperfusion. Analysis of vertebral bones was carried out as described in the Supplemental Experimental Procedures. Tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA) treatment was done as previously described (O'Leary et al., 2007O'Leary O.F. Bechtholt A.J. Crowley J.J. Hill T.E. Page M.E. Lucki I. Depletion of serotonin and catecholamines block the acute behavioral response to different classes of antidepressant drugs in the mouse tail suspension test.Psychopharmacology (Berl.). 2007; 192: 357-371Crossref PubMed Scopus (161) Google Scholar). Briefly, mice were treated with 200 mg/kg i.p. PCPA methyl ester hydrochloride (Sigma) or water twice daily (at 9:00 am and 4:00 pm) for 3 days (n = 44). Mice and food were weighed daily to ensure no adverse effect on body weight or appetite. On the fourth day, mice received mouse recombinant leptin (Merck, 5 mg/kg, i.p.) or vehicle 120 min and 30 min prior to the onset of the dark cycle (lights off at 7:00 pm) and food was removed. At the onset of the dark cycle, food was returned and subsequent 90 min intake was assessed. On completion of the study, animals were sacrificed (n = 11 per group) and brains rapidly removed and frozen. Brains were sectioned coronally at 150 μm on a cryostat and mounted onto glass slides. Micropunches were taken from the hypothalamus and striatum and homogenized in 60 μl of 0.2 M perchloric acid by an ultrasonic cell disruptor. Levels of 5-HT, 5-HIAA, DA, and NA in the supernatant were determined by HPLC and electrochemical detection, as described previously (Dalley et al., 2002Dalley J.W. Theobald D.E. Eagle D.M. Passetti F. Robbins T.W. Deficits in impulse control associated with tonically-elevated serotonergic function in rat prefrontal cortex.Neuropsychopharmacology. 2002; 26: 716-728Crossref PubMed Scopus (225) Google Scholar). Acute coronal brain slices (250 μm thick) containing the DR were prepared from 22- to 44-day-old mice and standard whole-cell patch-clamp recordings were performed (Burdakov and Ashcroft, 2002Burdakov D. Ashcroft F.M. Cholecystokinin tunes firing of an electrically distinct subset of arcuate nucleus neurons by activating A-Type potassium channels.J. Neurosci. 2002; 22: 6380-6387PubMed Google Scholar); see the Supplemental Experimental Procedures. As in previous studies (Yadav et al., 2009Yadav V.K. Oury F. Suda N. Liu Z.W. Gao X.B. Confavreux C. Klemenhagen K.C. Tanaka K.F. Gingrich J.A. Guo X.E. et al.A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure.Cell. 2009; 138: 976-989Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar), to facilitate the firing of serotonin neurons, we supplemented aCSF (see the Supplemental Experimental Procedures for details) with phenylephrine (3 μM, Tocris). The following synaptic blockers were also added to aCSF: AP5 (50 μM), CNQX (10 μM), picrotoxin (50 μM), strychnine (3 μM), and CGP52432 (10 μM) (Tocris). Data collection and analysis were performed using Pulse (HEKA). To visualize neurons after recordings, biocytin (0.5 mg/ml, Tocris) was added to the pipette solution (see the Supplemental Experimental Procedures for details), and the slices were fixed in 10% formalin. Immunohistochemical processing for the detection of 5-HT was performed as detailed above and images were acquired using an Olympus BX61WI confocal microscope. The effect of drug treatment on cell firing rate was assessed using t test. The effects of PCPA on 5-HT, 5-HIAA, DA, and NA levels in the striatum and hypothalamus were analyzed with t test. The effect of PCPA pretreatment and leptin treatment on food intake was assessed with a two-way ANOVA, followed by Tukey HSD post hoc test. The effect of genotype on body weight, gonadal fat, and bone density were analyzed with t test. For all analyses, significance was assigned at the p ≤ 0.05 level. This work was supported by grants from the Gates Cambridge Trust (D.D.L), the Wellcome Trust WT081713 (L.K.H), the Marilyn H. Vincent Foundation and American Diabetes Association (M.G.M.), NIH (HD061539 to C.E., DK065171 to L.K.H., DK057768 and DK078056 to M.G.M.), and the Cambridge Medical Research Council Centre for Study of Obesity and Related Disorders (L.K.H., D.I.B., and M.L.E.). We thank Drs. Xiaoxi Zhuang and Streamson Chua for the gift of Sertcre and Leprfl mice, respectively. We thank Amylin Pharmaceuticals for the generous gift of leptin. Core support was provided by NIH DK20572 and DK089503. Download .pdf (4.94 MB) Help with pdf files Document S1. Four Figures, Supplemental Experimental Procedures, and Supplemental References" @default.
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• W2107881559 title "Leptin Does Not Directly Affect CNS Serotonin Neurons to Influence Appetite" @default.
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