TrkB-expressing neurons in the dorsomedialhypothalamus are necessary and sufficientto suppress homeostatic feedingGuey-Ying Liaoa, Clint E. Kinneya, Juan Ji Ana, and Baoji Xua,1

aDepartment of Neuroscience, The Scripps Research Institute Florida, Jupiter, FL 33458

Edited by Richard D. Palmiter, University of Washington, Seattle, WA, and approved January 3, 2019 (received for review September 11, 2018)

Genetic evidence indicates that brain-derived neurotrophic factor(BDNF) signaling through the TrkB receptor plays a critical role inthe control of energy balance. Mutations in the BDNF or the TrkB-encoding NTRK2 gene have been found to cause severe obesity inhumans and mice. However, it remains unknownwhich brain neuronsexpress TrkB to control body weight. Here, we report that TrkB-expressing neurons in the dorsomedial hypothalamus (DMH) regulatefood intake. We found that the DMH contains both glutamatergic andGABAergic TrkB-expressing neurons, some of which also express theleptin receptor (LepR). As revealed by Fos immunohistochemistry, asignificant number of TrkB-expressing DMH (DMHTrkB) neurons wereactivated upon either overnight fasting or after refeeding. Chemoge-netic activation of DMHTrkB neurons strongly suppressed feeding inthe dark cycle when mice are physiologically hungry, whereas chemo-genetic inhibition of DMHTrkB neurons greatly promoted feeding in thelight cycle when mice are physiologically satiated, without affectingfeeding in the dark cycle. Neuronal tracing revealed that DMHTrkB

neurons do not innervate neurons expressing agouti-related proteinin the arcuate nucleus, indicating that DMHTrkB neurons are distinctfrom previously identified LepR-expressing GABAergic DMH neuronsthat suppress feeding. Furthermore, selective Ntrk2 deletion in theDMH of adult mice led to hyperphagia, reduced energy expenditure,and obesity. Thus, our data show that DMHTrkB neurons are a popu-lation of neurons that are necessary and sufficient to suppress appetiteand maintain physiological satiation. Pharmacological activation ofthese neurons could be a therapeutic intervention for the treatmentof obesity.

TrkB | dorsomedial hypothalamus | food intake | energy expenditure |obesity

Globally, 10.8% of men and 14.9% of women were obese in2014 (1). Prevalence of obesity is even higher in the United

States, where 34.9% of adults and 17% of youth were obese in2011–2012 (2, 3). Obese youth and adults develop type 2 diabetes athigh rates and are at significant risk for life-threatening cardiovas-cular disease and cancer (4, 5). So far, the most effective treatmentof severe obesity and diabetes is invasive bariatric surgeries to re-organize the gastrointestinal tract (6). Elucidation of the mechanismgoverning energy balance should provide an opportunity to developnovel effective and noninvasive therapeutic interventions.Brain-derived neurotrophic factor (BDNF) is well known for its

role in neuronal development and synaptic function (7). Geneticstudies show that BDNF is also crucial for the control of energybalance (8). Mutations that impair BDNF signaling through theTrkB receptor have been found to cause severe obesity in mice (9–12). Importantly, the BDNF gene has been associated with humanobesity in genome-wide association studies (13). Furthermore, likemutations in leptin and melanocortin-4 receptor, mutations in theBDNF or the TrkB-encoding NTRK2 gene lead to severe obesity inhumans (14–19). We and others have identified the paraventricularhypothalamus and the ventromedial hypothalamus (VMH) as twomain brain areas in which neurons produce BDNF to suppress foodintake and promote energy expenditure (20–22). However, the

anatomical TrkB-expressing site where BDNF acts to regulate en-ergy balance remains unknown.In this study, we employed Cre-expressing adeno-associated

virus (AAV) to selectively delete the Ntrk2 gene in the dorso-medial hypothalamus (DMH) and designer receptors exclusivelyactivated by designer drugs (DREADDs) to alter the activity ofTrkB-expressing neurons in the DMH, and then examined theeffect of these genetic manipulations on food intake and bodyweight. Results from these experiments show that TrkB-expressing DMH (DMHTrkB) neurons are key regulatorsof appetite.

ResultsDMHTrkB Neurons Respond to Fasting and Refeeding. To identifyTrkB-expressing neurons in the DMH, we crossed Ntrk2CreER/+

mice (23) and Ai9 mice (24) to generate Ntrk2CreER/+;Ai9/+double-heterozygous mice, in which Cre-mediated excision of aloxP-flanked STOP cassette allows for transcription of a CAGpromoter-driven tdTomato in TrkB-expressing cells after ta-moxifen treatment. We detected many tdTomato-labeled TrkB-expressing cells in the arcuate nucleus (ARC), VMH, and DMH(Fig. 1 A–C). Both astrocytes and neurons in the hypothalamusexpress TrkB (11). To distinguish neurons from astrocytes, weperformed immunohistochemistry against the neuronal markerNeuN and the astrocytic marker glial fibrillary acidic proteinin Ntrk2CreER/+;Ai9/+ brain sections. We found that both theARC and DMH have TrkB-expressing astrocytes and neurons,

Significance

The dorsomedial hypothalamus (DMH) is well known for its role inthe regulation of energy expenditure; however, its role in thecontrol of appetite is less defined. Our study identifies TrkB-expressing DMH neurons that potently suppress appetite andefficiently maintain physiological satiety when they are activated.Furthermore, because ablating the TrkB receptor in these neuronsincreased appetite and reduced energy expenditure, our studyindicates that BDNF could act in part on the DMH to control bodyweight. These results suggest that activation of DMH TrkBneurons could be a powerful way to treat obesity because itwill not only greatly reduce appetite but also overcome a potentcounterregulatory mechanism by which food restriction-inducedweight loss disproportionately reduces energy expenditure.

Author contributions: G.-Y.L. and B.X. designed research; G.-Y.L., C.E.K., and J.J.A. per-formed research; G.-Y.L., J.J.A., and B.X. analyzed data; and G.-Y.L. and B.X. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence should be addressed. Email: bxu@scripps.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1815744116/-/DCSupplemental.

Published online February 4, 2019.

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whereas the majority of TrkB-expressing cells in the VMH areastrocytes (SI Appendix, Figs. S1 and S2). We focus on DMHTrkB

neurons in this study.TrkB-expressing neurons were found in all regions of the DMH

along anterior–posterior, dorsal–ventral, and lateral–medial axes(Fig. 1 D–I). It appears that density of TrkB-expressing neurons inthe ventral part of the DMH (vDMH) and central part of the DMHis higher than in the dorsal part of the DMH (Fig. 1 E and F).We then sought to investigate whether DMHTrkB neurons are

activated by energy deficit (fasting) or energy repletion (refeeding)by examining the expression of Fos, an indirect marker of neuronalactivity. Immunohistochemistry detected few Fos+ cells in the DMHof normally fed mice (SI Appendix, Fig. S3 A–C). Conversely, manyFos+ DMH cells were found in mice fasted overnight (SI Appendix,Fig. S3 D–F) or in mice that were refed for 2 h after overnightfasting (SI Appendix, Fig. S3 G–I). We found that a significantamount of Fos immunoreactivity in the DMH was in TrkB-expressing neurons under these two conditions (Fig. 2A). Quan-tification revealed that approximately one-fourth of DMHTrkB

neurons were activated by either fasting or refeeding (Fig. 2B).Because fasting and refeeding can alter hunger and thermo-genesis, these results suggest that DMHTrkB neurons could beinvolved in the regulation of appetite and energy expenditure.

Neurochemical Characterization of DMHTrkB Neurons. The DMHharbors both glutamatergic and GABAergic neurons, with thelatter as the dominant population (25). To determine whetherDMHTrkB neurons are glutamatergic or GABAergic, we crossedeither Slc17a6Cre/+ mice (also termed Vglut2-ires-Cre mice; aCre driver specific to glutamatergic neurons) or Slc32a1Cre/+

mice (also termed Vgat-ires-Cre mice; a Cre driver specific toGABAergic neurons) (25) to Ntrk2fBZ/+ mice in which Cre-mediated deletion of the Ntrk2 gene leads to production ofβ-galactosidase in TrkB-expressing cells (26). The resulting

Slc17a6Cre/+;Ntrk2fBZ/+ or Slc32a1Cre/+;Ntrk2fBZ/+ offspring willexpress β-galactosidase in glutamatergic or GABAergic TrkB-expressing neurons, respectively. We detected β-galactosidase-expressing neurons in both Slc17a6Cre/+;Ntrk2fBZ/+ and Slc32a1Cre/+;Ntrk2fBZ/+ mice (Fig. 3 A and B). While glutamatergic TrkB-expressing neurons are more concentrated in the central and lat-eral DMH (Fig. 3A), GABAergic TrkB-expressing neurons appearto be more abundant in the central DMH and vDMH (Fig. 3B).These results indicate that there are both glutamatergic andGABAergic populations of TrkB-expressing neurons in the DMH.To assess whether glutamatergic and GABAergic DMHTrkB

neurons would respond differently to fasting versus refeeding, weexamined the expression of Fos in glutamatergic or GABAergicDMHTrkB neurons under these two conditions. Fos expressionwas detected in both glutamatergic and GABAergic DMHTrkB

neurons of fasted mice (SI Appendix, Figs. S4 and S5). Similarly,a fraction of both glutamatergic and GABAergic DMHTrkB

neurons was activated in refed mice (SI Appendix, Figs. S4 andS5). Thus, both populations of glutamatergic and GABAergicDMHTrkB neurons are involved in the fasting- or refeeding-induced neuronal activation.Leptin is a hormone crucial to energy balance and acts through

the leptin receptor (LepR) expressed in multiple brain regions, in-cluding the DMH (27). Recently, LepR-expressing GABAergicneurons in the vDMH have been found to suppress feeding byinhibiting ARC neurons that express agouti-related protein (AgRP)(28). In addition, LepR-expressing neurons in the DMH/dorsalhypothalamic area have been shown to play a role in the control ofenergy expenditure and body weight (29). To determine whethersome of the DMHTrkB neurons respond to leptin, we administeredleptin into Ntrk2CreER/+;Ai9/+ mice and then performed immuno-histochemistry to detect the immunoreactivity for phosphorylatedsignal transducers and activators of transcription protein 3(pSTAT3), which has been shown to sensitively and reliably identify

Fig. 1. TrkB expression in the DMH. (A–C) Distribution of tdTomato-labeledTrkB-expressing cells in the ARC, DMH, lateral hypothalamus (LH), and VMH ofadultNtrk2CreER/+;Ai9/+mice. (Scale bar, 100 μm.) (D–F) Distribution of tdTomato-labeled TrkB-expressing cells in the DMH of adult Ntrk2CreER/+;Ai9/+ mice.(Scale bar, 100 μm.) (G–I) DAPI staining showing the cytoarchitecture of theDMH. dDMH, dorsal division of the DMH; DMC, central division of the DMH;vDMH, ventral division of the DMH.

Fig. 2. Activation of DMHTrkB neurons by fasting and refeeding. (A) In-duction of Fos in TrkB-expressing neurons in the DMH of fasted and refedNtrk2CreER/+;Ai9/+ mice. TrkB-expressing neurons were marked by tdTomatoin tamoxifen-treated Ntrk2CreER/+;Ai9/+ mice. Arrows denote neurons thatare positive for both Fos and TrkB. (Scale bar, 50 μm.) (B) Quantification ofneurons positive for both Fos and TrkB. n = 3 mice for each condition; **P <0.01 by Student’s t test.

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LepR-expressing neurons (30). In agreement with a previous report(27), we detected pSTAT3-positive neurons in the ARC, DMH,VMH, and lateral hypothalamus (Fig. 3C, C1, C5, and C9). How-ever, only 17% (83/477) of DMHTrkB neurons were immunoreactiveto pSTAT3 (Fig. 3C, C2–C4, C6–C8, and C10–C12). Therefore, themajority of DMHTrkB neurons do not respond to leptin.It has been reported that there are cholinergic neurons in the

DMH (31, 32) and that chemogenetic activation of these neuronspromotes food intake (33). We sought to determine whether someDMHTrkB neurons, especially those that were activated after over-night fasting, are cholinergic. We detected neurons expressingcholine acetyltransferase in the posterior hypothalamus, but not inthe DMH (SI Appendix, Fig. S6). Even in the posterior hypothala-mus, we did not see neurons that express both choline acetyl-transferase and TrkB (SI Appendix, Fig. S6). Therefore, DMHTrkB

neurons are not cholinergic.

Chemogenetic Modification of DMHTrkB Neuronal Activity Alters FoodIntake. Some DMHTrkB neurons were activated upon refeedingafter overnight fasting (Fig. 2), suggesting that these neuronsinhibit appetite. To test this possibility, we investigated the im-pact of activating DMHTrkB neurons on food intake using thestimulatory DREADD hM3D(Gq) and its ligand clozapine (34).We expressed the hM3D(Gq)-mCherry or control mCherryin DMHTrkB neurons by injecting Cre-dependent AAV-hSyn-DIO-hM3D(Gq)-mCherry or AAV-hSyn-DIO-mCherry into theDMH of Ntrk2CreER/+ mice and subsequently treating themice with tamoxifen (Fig. 4A). We found that administration of

clozapine N-oxide (CNO) drastically reduced food intake by82% over the first 2 h in Ntrk2CreER/+ mice expressing hM3D(Gq)-mCherry, but not in control mice expressing mCherry,during the dark cycle when mice are physiologically hungry andingest the vast majority of their daily energy intake, comparedwith vehicle administration (Fig. 4B). This result indicates thatactivation of DMHTrkB neurons is sufficient to suppress appetite.We then determined whether DMHTrkB neuronal activity is nec-

essary to maintain physiological satiation during the light cycle whenmice do not eat much food. We injected AAV-hSyn-DIO-hM4D(Gi)-mCherry (35) into Ntrk2CreER/+ mice to express the inhibitoryDREADD in DMHTrkB neurons. Administration of CNO greatlyincreased food intake by sixfold over the first 2 h inNtrk2CreER/+ miceexpressing hM4D(Gi)-mCherry, but not in mCherry-expressingcontrol mice, during the light cycle, compared with vehicle admin-istration (Fig. 4C). This result indicates that DMHTrkB neurons areactive to maintain physiological satiation during the light cycle. In-terestingly, we found that inhibition of DMHTrkB neurons withhM4D(Gi)/CNO did not increase food intake during the dark cycle(Fig. 4D), suggesting that the anorexigenic DMHTrkB neurons arefully inhibited when mice are physiologically hungry.Together, these chemogenetic experiments indicate that

DMHTrkB neurons play a critical role in the regulation of daily foodintake. Our data suggest that these neurons are silenced to allow forfeeding during the dark cycle and are active to suppress feeding andmaintain physiological satiation during the light cycle.

Fig. 3. Identity of TrkB-expressing neurons in the DMH. (A and B) Immunohistochemistry of β-galactosidase (β-gal) reveals glutamatergic TrkB-expressingDMH neurons in Slc17a6Cre/+;Ntrk2fBZ/+ mice (A) and reveals GABAergic TrkB-expressing DMH neurons in Slc32a1Cre/+;Ntrk2fBZ/+ mice (B). DAPI staining displaysthe cytoarchitecture of the DMH. (Scale bar, 50 μm.) (C) Immunoreactivity of pSTAT3 was induced in tdTomato-labeled TrkB-expressing DMH neurons inleptin- and tamoxifen-treated Ntrk2CreER/+;Ai9/+ mice. Arrows denote DMHTrkB neurons that are immunoreactive to pSTAT3. (Scale bars, 100 μm.)

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DMHTrkB Neurons Project to the ARC, but Not onto Either AgRP orProopiomelanocortin Neurons. Given that some DMHTrkB neuronsare GABAergic and express LepR, we asked whether feeding-suppressing DMHTrkB neurons could be the same as the pre-viously identified GABAergic vDMHLepR neurons that innervateand inhibit AgRP neurons to suppress food intake (28). To thisend, we first determined whether DMHTrkB neurons project to theARC by injecting Cre-dependent AAV-FLEX-tdTomato into theDMH ofNtrk2CreER/+ mice. After tamoxifen treatment, we observedaxonal terminals of tdTomato-expressing DMHTrkB neurons in theARC (Fig. 4E), indicating that the ARC is a projection target ofDMHTrkB neurons. We next investigated whether DMHTrkB neu-rons innervate AgRP neurons by injecting the EnvA-pseudotypedmodified rabies virus SADΔG-EGFP into the ARC of AgrpCre/+;RΦGT/+;Ntrk2LacZ/+ mice (Fig. 4F). The RΦGT mice express therecombinant rabies glycoprotein (RABVgp4) and the receptor forEnvA, TVA (36). Injected SADΔG-EGFP virus would be taken upby ARCAgRP neurons through TVA and transported retrogradely toafferent neurons after replication in the presence of RABVgp4. Wedetected many EGFP-expressing ARCAgRP afferent neurons in theDMH (Fig. 4F). These EGFP+ neurons were distinct fromβ-galactosidase–expressing TrkB neurons (Fig. 4G). Therefore,DMHTrkB neurons do not innervate ARCAgRP neurons and aredistinct from vDMHLepR neurons.We next employed the same neuronal tracing method to inves-

tigate whether DMHTrkB neurons innervate well-studied anorexi-genic proopiomelanocortin (POMC)-expressing neurons in theARC. Using PomcCre/+;RΦGT/+;Ntrk2LacZ/+ mice and rabies virusSADΔG-EGFP, we found that some DMH neurons did inner-vate ARCPOMC neurons (SI Appendix, Fig. S7A); however, none ofthese EGFP-expressing DMH neurons was a TrkB neuron marked

by β-galactosidase (SI Appendix, Fig. S7B). Thus, DMHTrkB neuronsdo not directly activate ARCPOMC neurons to suppress food intake.

Deletion of the Ntrk2 Gene in the DMH Leads to Obesity. Given thatBDNF is a potent modulator of synaptic function (7), we rea-soned that BDNF could regulate energy balance by modulatingthe activity of DMHTrkB neurons through the TrkB receptor. Totest this hypothesis, we investigated the impact of deleting theNtrk2 gene in the DMH on energy balance. We stereotaxicallyinjected AAV-GFP or AAV-Cre-GFP into the DMH of 8-wk-old female Ntrk2lox/lox mice bilaterally (Fig. 5A). AAV-Cre-GFPinjection abolished Ntrk2 gene expression in the DMH (SI Ap-pendix, Fig. S8A) and led to obesity (Fig. 5 B and C) in associ-ation with impaired glucose tolerance (Fig. 5E). Deletion of theNtrk2 gene in the DMH also led to increased weight gain in malemice (SI Appendix, Fig. S8B). Surprisingly, as does a germlineBdnf or Ntrk2 mutation (10, 12), Ntrk2 deletion in the matureDMH still led to increased body length (Fig. 5D), which could bethe main reason for increased lean mass (Fig. 5C). This resultsuggests that impairment in TrkB signaling still promotes lineargrowth, even after development is completed. Therefore, ourresults indicate that the TrkB receptor in the DMH plays a rolein the control of energy balance.Deletion of the Ntrk2 gene in the DMH increased daily food

intake (Fig. 5F), which further supports the role of DMHTrkB

neurons in suppression of appetite and maintenance of satiation.In addition to hyperphagia, reduced energy expenditure con-tributed to the obesity phenotype observed in mice in whichNtrk2 was deleted in the DMH, because indirect calorimetryrevealed that Ntrk2 deletion in the DMH reduced oxygen con-sumption per animal (Fig. 5G) or normalized to lean mass (Fig. 5H).

Fig. 4. Chemogenetic modification of DMHTrkB neuronal activity and food intake. (A) Bilateral injection of AAV-hSyn-DIO-hM3D(Gq)-mCherry into the DMH ofNtrk2CreER/+ mice. The brain section was counterstained with DAPI. (Scale bar, 100 μm.) (B) Activation of DMHTrkB neurons suppressed food intake during a nocturnalphase (active period). n = 10 mice in the AAV-hSyn-DIO-hM3D(Gq)-mCherry (hM3) group and n = 7mice in the AAV-hSyn-DIO-mCherry (control, Ctrl) group. Repeatedmeasures two-way ANOVA for effect of treatment [F(3, 120) = 37.2, P < 0.0001] and Bonferroni post hoc test [**P < 0.01 and ***P < 0.001 compared with the control-vehicle (Ctrl-Veh) group]. (C) Inhibition of DMHTrkB neurons stimulated food intake during a diurnal phase (inactive period). n = 7mice in the AAV-hSyn-DIO-hM4D(Gi)-mCherry (hM4) group and n = 7 mice in the AAV-hSyn-DIO-mCherry (control) group. Repeated measures two-way ANOVA for effect of treatment [F(3, 96) = 44.2, P <0.0001] and Bonferroni post hoc test (**P < 0.01 and ***P < 0.001 compared with the control-vehicle group). (D) Inhibition of DMHTrkB neurons did not change foodintake during a nocturnal phase. n = 4 mice in the AAV-hSyn-DIO-hM4D(Gi)-mCherry (hM4) group and n = 7 mice in the AAV-hSyn-DIO-mCherry (control) group.Repeated measures two-way ANOVA for effect of treatment; F(3, 72) = 1.34, P = 0.2691. (E) Unilateral injection of AAV-CAG-FLEX-tdTomato into the DMH ofNtrk2CreER/+ mice revealed projection of DMHTrkB neurons to the ARC (E′, Inset and enlargement). (Scale bar, 50 μm.) (F) Unilateral injection of SADΔG-EGFP into theARC of ArgpCre/+;RΦGT/+;Ntrk2LacZ/+ mice revealed ARCAgRP afferent neurons in the DMH. (Scale bar, 50 μm.) (G) DMHTrkB neurons, marked by β-galactosidase (β-gal)immunoreactivity, were not transduced by SADΔG-EGFP virus. (Scale bar, 50 μm.)

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The reduction in energy expenditure was attributable in partto decreased ambulatory activity in the Ntrk2 mutant mice(Fig. 5I). These results suggest that DMH TrkB is involved inboth suppression of appetite as well as promotion of energyexpenditure.

DiscussionIn this study, we identify DMHTrkB neurons as key regulators ofenergy balance and body weight by means of Fos immunohis-tochemistry, chemogenetic neuronal activation or inhibition, andselective deletion of the Ntrk2 gene. Our results also suggest thatDMHTrkB neurons may participate in the control of daily in-gestion behavior influenced by the day/night cycle.Our data show that there are both glutamatergic and GABAergic

TrkB-expressing neurons in the DMH. We found that DREADD-mediated activation of DMHTrkB neurons suppressed feeding dur-ing the dark cycle, whereas DREADD-mediated silencing of theseneurons stimulated feeding during the light cycle. This observationindicates that these appetite-controlling DMHTrkB neurons arelikely distinct from GABAergic vDMHLepR neurons that innervateARCAgRP neurons. Chemogenetic silencing of vDMHLepR neuronsdoes not increase food intake during the light cycle, although acti-vation of these neurons does suppress feeding by inhibiting AgRPneurons in the ARC (28). Furthermore, our neuronal tracing resultsshow that DMHTrkB neurons do not innervate ARCAgRP neurons.Additionally, our finding that only a small fraction of DMHTrkB

neurons was responsive to leptin further supports the view thatthese two groups of appetite-controlling neurons are distinct. It isimportant to identify in future studies the postsynaptic targetthrough which DMHTrkB neurons potently modulate appetite.It is unlikely that all DMHTrkB neurons are involved in the

control of feeding. We found that overnight fasting activated∼25% of DMHTrkB neurons. Given that chemogenetic activationof DMHTrkB neurons nearly halted food intake during the darkcycle, these fasting-activated DMHTrkB neurons may not have anorexigenic role. Instead, these neurons may be activated in re-sponse to other fasting-induced physiological changes, such asreduced adaptive thermogenesis (20, 37). Indeed, both cold andwarm temperatures have been found to activate DMH neurons

to alter adaptive thermogenesis (38). In support of this expla-nation, we found that Ntrk2 deletion in the DMH reduced energyexpenditure. In other words, DMHTrkB neurons that are activatedby fasting could be involved in thermoregulation, and they may beactivated in an attempt to increase adaptive thermogenesis in re-sponse to fasting-induced reduction in body temperature (39).However, it is important to note that DREADD-mediated activa-tion of DMHTrkB neurons represents a supraphysiological stimula-tion. Nevertheless, we found that deleting the Ntrk2 gene in theDMH reduced energy expenditure, which is in line with the possi-bility that fasting-activated DMHTrkB neurons are involved inthermoregulation. Because BDNF-expressing neurons in the para-ventricular hypothalamus and preoptic area are important for theregulation of thermogenesis (20, 40), it would be interesting to de-termine in future investigations whether these BDNF neurons are inthe same circuit as DMHTrkB neurons to control thermogenesis.Several studies have demonstrated that the DMH is a critical

hypothalamic nucleus for food-entrainable circadian rhythms (41–43). We made an interesting observation that silencing of DMHTrkB

neurons increased food intake during the light cycle withoutaffecting food intake during the dark cycle. This suggests thatDMHTrkB neurons likely are active during the light cycle to inhibitfeeding and are silenced during the dark cycle to allow feeding. Thisinference is further supported by the finding that forced activationof DMHTrkB neurons using hM3D(q) almost halted feeding duringthe dark cycle. Definitive evidence regarding the diurnal change inthe activity of DMHTrkB neurons will come from electrophysiolog-ical recordings once these appetite-controlling DMHTrkB neuronsare molecularly defined. If our inference is true, DMHTrkB neuronsshould play a critical role in regulating light/dark-entrained circa-dian rhythms of hunger and satiation. It would be intriguing to in-vestigate how the suprachiasmatic nucleus (the circadian clock)regulates the activity of DMHTrkB neurons, since DMH is a pro-jection target of the suprachiasmatic nucleus (44).As BDNF is a potent regulator of synaptic function (7), it is likely

that BDNF regulates feeding in part by modulating the activity ofDMHTrkB neurons. For example, afferents to DMHTrkB neuronscould be BDNF-expressing neurons, and BDNF may enhancesynaptic transmission at their connections. Given that Ntrk2

Fig. 5. Deletion of the Ntrk2 gene in the adult DMH using Cre-expressing AAV. (A) Stereotaxic injection of AAV-GFP or AAV-Cre-GFP into the DMH ofNtrk2lox/lox mice. (Scale bar, 100 μm.) (B) Body weight of female Ntrk2lox/lox mice injected with either AAV-GFP (GFP) or AAV-Cre-GFP (Cre). n = 6 and 8 mice forGFP and Cre groups, respectively. Two-way ANOVA with Bonferroni post hoc test; F(1, 96) = 101, P < 0.0001 for viral injection; *P < 0.05, **P < 0.01, and ***P <0.001. (C) Body composition measured at 5 wk after viral injection. Student’s t test; **P < 0.01 and ***P < 0.001. (D) Body length. Student’s t test; ***P <0.001. (E) Glucose tolerance test. Two-way ANOVA with Bonferroni post hoc test; F(1, 70) = 13.7, P < 0.0001 for viral injection; *P < 0.05 and **P < 0.01. (F) Dailyfood intake. Student’s t test; *P < 0.05. (G) Oxygen consumption per animal, measured using comprehensive laboratory animal monitoring system. Studentt test; *P < 0.05. (H) Oxygen consumption normalized to lean mass. Student’s t test; *P < 0.05. (I) Ambulatory activity, measured using comprehensivelaboratory animal monitoring system. Two-way ANOVA with Bonferroni post hoc test; F(1, 288) = 21.85 and P < 0.0001 for viral injection; **P < 0.01.

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deletion in the DMH increases food intake and reduces energyexpenditure, DMHTrkB neurons could be a good drug target forobesity treatment. Because food restriction-induced weight lossdisproportionately reduces energy expenditure as a powerfulcounter regulatory mechanism (45), effective reversal of obesityrequires a combination of pharmacotherapies to inhibit food intakeand increase energy expenditure. Pharmacological activation ofDMHTrkB neurons would suppress appetite, induce weight loss, andprevent the associated reduction in energy expenditure.

Materials and MethodsAnimals. Floxed Ntrk2 (Ntrk2lox; also known as TrkBlox), floxed Ntrk2-LacZ(Ntrk2fBZ; also known as fBZ), and Ntrk2LacZ (also known as TrkBLacZ)mouse strains were previously described (46, 47). Ai9 [Gt(ROSA)26Sortm9

(CAG-tdTomato)Hze/J; stock no. 007909], Slc32a1Cre (Vgat-ires-Cre; stock no.016962), Slc17a6Cre (Vglut2-ires-Cre; stock no. 028863), Gad2Cre (stock no.028867), RΦGT [Gt(ROSA)26Sortm1(CAG-RABVgp4,-TVA)Arenk/J; stock no. 024708],PomcCre (stock no. 010714), and AgrpCre (stock no. 012899) mouse strainswere obtained from The Jackson Laboratory. The Ntrk2CreER/+ (also known asTrkBCreER) mouse strain (23) was kindly provided by David Ginty, HarvardMedical School, Boston. Details on animal housing and treatment areavailable in SI Appendix, SI Materials and Methods. The Animal Care and UseCommittees at The Scripps Research Institute Florida approved all animalprocedures used in this study.

Viral Injection. AAVs were stereotaxically injected into the DMH or ARC of8-wk-old mice. Mice were singly housed for 1 wk after surgery to facilitaterecovery and then group housed. For virus-injected Ntrk2CreER/+ mice, ta-moxifen was administered 1 wk after surgery, and experiments were con-ducted 2 wk afterward. Details are available in SI Appendix, SI Materialsand Methods.

Immunohistochemistry. Series of 40-μm coronal brain sections were used forimmunohistochemistry. Images were acquired using a Nikon C2+ confocalmicroscope. Colocalization was determined on confocal images. Furtherdetails are available in SI Appendix, SI Materials and Methods.

Physiological Measurement. Food intake was measured in mice expressinghM3Dq or hM4Di during the light or dark cycle after saline or CNO injection.Body weight, daily food intake, energy expenditure, ambulatory activity, andglucose tolerance were monitored in control and Ntrk2 mutant mice. Detailsare available in SI Appendix, SI Materials and Methods.

Statistical Analyses. Statistical significance was determined using GraphPadPrism or Excel software. P < 0.05 was considered significant. All data areexpressed as mean ± SEM.

ACKNOWLEDGMENTS. We thank Dr. David Ginty for the Ntrk2CreER mousestrain and Jessica Houtz, Shaw-wen Wu, and Xiangyang Xie for critical read-ing of this manuscript. This work was supported by National Institutes ofHealth Grants R01 DK105954 and R01 DK103335 (to B.X.).

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