Multinodal regulation of the arcuate/paraventricularnucleus circuit by leptinMasoud Ghamari-Langroudia,1, Dollada Srisaib, and Roger D. Conea,1

aDepartment of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-0615; and bDepartment of Anatomy,Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand

Contributed by Roger D. Cone, November 19, 2010 (sent for review September 14, 2010)

Melanocortin-4 receptor (MC4R) is critical for energy homeostasis,and the paraventricular nucleus of the hypothalamus (PVN) isa key site of MC4R action. Most studies suggest that leptinregulates PVN neurons indirectly, by binding to receptors in thearcuate nucleus or ventromedial hypothalamus and regulatingrelease of products like α-melanocyte-stimulating hormone(α-MSH), neuropeptide Y (NPY), glutamate, and GABA from first-order neurons onto the MC4R PVN cells. Here, we investigatemechanisms underlying regulation of activity of these neurons un-der various metabolic states by using hypothalamic slices froma transgenic MC4R-GFP mouse to record directly from MC4R neu-rons. First, we show that in vivo leptin levels regulate the tonicfiring rate of second-order MC4R PVN neurons, with fasting in-creasing firing frequency in a leptin-dependent manner. We alsoshow that, although leptin inhibits these neurons directly at thepostsynaptic membrane, α-MSH and NPY potently stimulate andinhibit the cells, respectively. Thus, in contrast with the conven-tional model of leptin action, the primary control of MC4R PVNneurons is unlikely to be mediated by leptin action on arcuateNPY/agouti-related protein and proopiomelanocortin neurons.We also show that the activity of MC4R PVN neurons is controlledby the constitutive activity of the MC4R and that expression of thereceptor mRNA and α-MSH sensitivity are both stimulated by lep-tin. Thus, leptin acts multinodally on arcuate nucleus/PVN circuitsto regulate energy homeostasis, with prominent mechanisms in-volving direct control of both membrane conductances and geneexpression in the MC4R PVN neuron.

melanocortin signaling | electrophysiology | obesity

Neurons expressing melanocortin-4 receptor (MC4R) in theparaventricular nucleus of the hypothalamus (PVN) play

a crucial role in energy homeostasis. The genetic and pharma-cological disruption of MC4R increases energy intake anddecreases thermogenesis (1, 2). Thus, these neurons sense pe-ripheral signals of adiposity and maintain energy homeostasis bycoordinating energy intake and expenditure (2, 3). The adipocytehormone leptin relays information on changes in peripheralenergy stores to melanocortin and other circuits in the brain toregulate energy homeostasis (4). Neurons in the arcuate nucleusof the hypothalamus (ARC) and other nuclei, including theventromedial nucleus (VMH), dorsomedial nucleus, and lateralnucleus, express leptin receptors and play an important role intransmitting the leptin signal to PVN neurons (5–7). Despitefunctional evidence suggesting expression of the leptin receptorin PVN (8), direct action of leptin on PVN neurons has not beenthoroughly investigated, perhaps because of the low density ofleptin-receptor expression (9, 10).Circulating leptin, by affecting the activity of neuropeptide Y

(NPY) and proopiomelanocortin (POMC) neurons in the ARC,regulates synthesis of NPY/agouti-related protein (AgRP) andα-melanocyte-stimulating hormone (α-MSH) as well as their pu-tative release from nerve endings onto neurons in PVN (11, 12).The synthesis and release of inhibitory products of NPY/AgRPneurons—NPY, AgRP, and GABA—are suppressed by leptin(13), and the products of ARC POMC neurons then maintain anexcitatory effect on PVN neurons (14). Removal of that excit-atory effect pharmacologically, or by destruction of ARC neurons

(15, 16), presumably decreases the activity of PVN neurons,resulting in increases in energy intake. Despite advances in ourunderstanding of the regulation of ARC POMC and NPY/AgRPneurons, mechanisms involved in regulation of downstream ef-fector PVN neurons are less well understood. Recent studies haveshown that deletion of leptin receptors on ARC POMC neuronsonly partially reduces the extremeobesity seenwith global deletionof leptin-receptor signaling (17), suggesting involvement of otherhypothalamic nuclei in relaying the peripheral signals of adiposity.In fact, deletion of leptin receptors inVMHneurons also increasesbody weight (18), further underscoring a role of leptin-receptorsignaling in “non-ARC” neuronal centers. These studies thussuggest involvement of multiple neuronal centers in sensing andrelaying information of energy status to theCNS (19). In this study,we investigate mechanisms involved in regulation of downstreameffector PVN neurons in vivo by analyzing how metabolic state,leptin, and products of ARC neurons regulate the activity ofMC4R effector neurons in PVN.

ResultsMC4R PVN Neurons Are Tonically Regulated by Metabolic State. Wesought to understand how the activity of MC4R PVN effectorneurons is regulated in different metabolic states by recordingfrom cells expressing GFP under the control of the MC4Rpromoter (20), primarily in the mid- to posterior region of themouse PVN. We first measured action-potential firing frequencyof these neurons in hypothalamic slices obtained from mice ei-ther fed ad libitum or fasted (16 h). Using loose-patch record-ings, we obtained results from 23 mice that indicate that fastingsignificantly increased mean frequency of action-potential firingof these neurons compared with the ad libitum–fed state (2.7 ±0.2 Hz in fasted mice, n= 134, 1.9 ± 0.2 Hz in fed mice, n= 116,P < 0.005, unpaired t test, Fig. 1A).We next tested whether leptin mediated the fasting-induced

increase in activity of MC4R PVN neurons by comparing thefiring activity of neurons from fasted mice administered 3 mg/kgleptin i.p. or the same volume of saline (12). Using loose-patchrecording to sample firing frequency in MC4R PVN neuronsfrom 18 mice, we found that leptin suppressed the activity ofMC4R PVN neurons to ad libitum–fed levels (2.4 ± 0.2 Hz insaline-injected mice, n = 100, to 1.6 ± 0.2 Hz in leptin-injectedmice, n = 99, P < 0.005, unpaired t test, Fig. 1B). Frequencyanalysis indicates that fasting activates a subset of MC4R neu-rons firing at frequencies between 2.5 and 5.5 Hz, and leptinreplacement reversed the fasting-induced increase in this subsetof neurons (Fig. 1 Ai and Bii). To confirm that the fasting-in-duced activation of MC4R PVN neurons was mediated by en-dogenous leptin, we examined the firing activity of these neuronsin leptin-deficient ob/ob mice. We generated double-mutant

Author contributions: M.G.-L. and R.D.C. designed research; M.G.-L. and D.S. performedresearch; M.G.-L. and D.S. contributed new reagents/analytic tools; M.G.-L. and R.D.C.analyzed data; and M.G.-L. and R.D.C. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.1To whom correspondence may be addressed. E-mail: masoud.ghamari-langroudi@vanderbilt.edu or roger.cone@vanderbilt.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1016785108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1016785108 PNAS | January 4, 2011 | vol. 108 | no. 1 | 355–360

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MC4R-GFP ob/ob mice and then compared firing activity ofMC4R PVN neurons in these animals, fasted or fed ad libitum.The mean firing frequency of neurons obtained from fed MC4R-GFP × ob/ob mice was not significantly different from that of 16-h fasted MC4R-GFP × ob/ob mice (3.9 ± 0.2 Hz in fed, n = 132;3.8 ± 0.2 Hz in fasted mice, n = 105; unpaired t test, P > 0.5).The firing frequency of MC4R PVN neurons significantly de-creased when fasted MC4R-GFP × ob/ob mice were injectedwith leptin (3.8 ± 0.2 Hz in fasted mice, n = 105, to 2.1 ± 0.2 Hzin fasted, leptin-injected mice, n = 100, P < 0.0001, unpairedt test, Fig. 1C).

α-MSH Augments and AgRP Decreases Firing Activity of MC4R PVNNeurons Through MC4R Signaling. Leptin has been shown to affectneuronal activity of NPY/AgRP and POMC neurons in ARC andto regulate the release of their products (5, 11, 12, 21). To studythe actions of these ARC peptides on MC4R PVN neurons, wefirst compared average firing frequencies measured over 4–6 minin individual neurons in control conditions with frequencies ob-served in the presence of the melanocortin agonist MTII. Resultsobtained from 11 neurons (Fig. S1) indicate that bath applicationof 100 nM MTII significantly increased the firing rate in allneurons tested (1.4 ± 0.2 Hz in control conditions to 3.2 ± 0.5,n = 11 neurons from 11 mice, P < 0.001), in agreement withother reports of effects of MTII on PVN or ARC neurons(20, 22). Furthermore, this effect of MTII was abolished whenneurons were pretreated with an MC4R antagonist, 100 nMSHU9119 (23) (1.7 ± 0.9 Hz in SHU9119 alone to 1.8 ± 0.7 Hz inSHU9119 + MTII, P > 0.05, n = 7, Fig. S1).We then investigated the effects onfiring rate of the endogenous

agonist, α-MSH, and the endogenous inverse agonist, AgRP, ofMC4R signaling. Using loose-patch recordings, we tested theeffects of 250 nM α-MSH (24) on MC4R PVN neurons. In 13neurons tested, bath applications of α-MSH significantly and re-versibly increased by 78% the frequency of action-potential firing(from 1.7 ± 0.4 Hz in control conditions to 3.1 ± 0.5 Hz withα-MSH treatment, n = 13, P < 0.001, Fig. 2 A and B). Measured∼30–40 min after wash out, the firing frequency of MC4R PVNneurons was significantly reduced to values different from thoseobtained in the presence of α-MSH (2.1 ± 0.6, n = 13, P < 0.01).Using loose patch recording in seven MC4R PVN neurons, bathapplication of 100 nM AgRP significantly decreased (to 53% ofcontrol) the frequency of action-potential firing (from 2.7± 0.4Hzin control to 1.4± 0.5HzwithAgRP treatment,n=7,P<0.01, Fig.2 C and D).We next investigated whether the action of α-MSH is associ-

ated with changes in membrane potential of PVN neurons byusing whole-cell recordings. The membrane potential of PVNneurons was held near threshold for action potentials (25) be-tween −55 and −50 mV by injecting −20–0 pA of constant DCcurrent, allowing cells to fire action potentials spontaneously.The firing frequency and membrane potential of these neuronswere then monitored for 20–30 min under control conditions,before bath application of 250 nM α-MSH for 7–12 min, fol-

lowed by a longer period of wash out (>20 min). Firing frequencyand membrane potential were compared for a period of 4–6 minin control and for an identical period during α-MSH addition.Our results obtained from 24 MC4R PVN neurons tested underthese conditions indicate that bath application of 250 nM α-MSHsignificantly increased firing frequency in 21 of 24 neurons (from0.9 ± 0.2 Hz to 3.0 ± 0.6 Hz, n = 21, P < 0.0001, Fig. S2 A–D).Furthermore, our results indicate that 23 of 24 PVN cells ex-amined were depolarized by α-MSH. When all neurons wereincluded, α-MSH induced significant depolarization of mem-brane potential (from −54.2 ± 1.1 mV to −46.3 ± 0.9 mV, n= 24,P < 0.0001, Fig. S2 A–C and E).

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Fig. 1. Leptin tonically inhibits firing of MC4R PVN neurons. (A) Average ± SEM of action potential, firing frequency, and frequency distribution (i) of MC4RPVN neurons obtained from mice subjected to 16 h of fasting (n = 134) or fed ad libitum (n = 116, *P < 0.005). (B) Average ± SEM of firing frequency andfrequency distribution (ii) of these neurons from 16-h fasted mice that were injected i.p. 3 h before decapitation with 3 μg/g leptin (n = 100) or saline (n = 99,*P < 0.005). (C) Average ± SEM of frequency of firing of MC4R PVN neurons obtained from ob/obmice that were subjected to 16 h of fasting (n = 105), fed adlibitum (n = 132, P > 0.5), or injected i.p. with leptin 3 h before decapitation after 16 h of fasting (n = 100, *P < 0.0001).

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Fig. 2. α-MSH augments and AgRP and NPY decrease firing activity of MC4RPVN neurons. (A) Bath application of 250 nM α-MSH augments firing fre-quency of MC4R PVN neurons recorded using the loose-patch technique.(B) Average ± SEM of effect of 250 nM concentration of this peptideobtained from 13 MC4R PVN neurons (*P < 0.001). (C) Bath application of100 nM AgRP significantly inhibits firing frequency of PVN neurons obtainedby loose-patch recordings. (D) Average ± SEM of effect of 100 nM AgRP fromseven PVN neurons (*P < 0.01). (E) A whole-cell recording from a sponta-neously firing PVN neuron indicates that application of 100 nM NPY gen-erates a significant inhibition of firing activity associated with hyperpo-larization of membrane potential. (F and G) Average ± SEM of effect of100 nM NPY on firing frequency (*P < 0.05) and membrane potentials (*P <0.001) of eight MC4R PVN neurons.

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We also examined whether effects of α-MSH persisted in theabsence of GABA(A) and ionotropic glutamate neurotransmis-sion recorded from hypothalamic slices pretreated with 200 μMpicrotoxin (PTX) and 1 mM kynurenic acid (KYN) (26). Ap-plication of 250 nM α-MSH significantly increased firing fre-quency in six MC4R PVN neurons under these conditions (from1.2 ± 0.1 Hz to 2.7 ± 0.6 Hz, P < 0.05, n = 6, Fig. S2 F and G).This increase in firing activity was associated with a significantdepolarization of membrane potentials (from −55.4 ± 1.1 mV to−49.2 ± 2.1 mV, P < 0.005, n = 9, Fig. S2H). These findings areconsistent with α-MSH acting independently of ionotropicGABAergic or glutamatergic neurotransmission to depolarizeMC4R PVN neurons. However, they do not exclude a role ofother neurotransmitters in mediating the observed effects ofα-MSH (27). We hence tested effects of bath applications ofα-MSH on membrane potential of PVN neurons, as describedabove, in the absence of all action potential–dependent synaptictransmission by recording from hypothalamic slices pretreatedwith 0.5 μM tetrodotoxin. Application of α-MSH significantlydepolarized membrane potential of PVN neurons pretreatedwith tetrodotoxin (from −56.8 ± 1.1 mV to −50.7 ± 2.1 mV, n= 6,P < 0.01, Fig. S2H).

NPY Inhibits Firing in MC4R PVN Neurons. Because NPY neurons inARC project to MC4R PVN neurons, where Y receptors havebeen found in abundance (28), we also examined effects of NPY.Using whole-cell recordings, we examined effects of this peptideon firing frequency as well as membrane potentials of MC4RPVN neurons by comparing these parameters in control solutionand in the presence of 100 nM NPY. Bath application of NPYconsistently and reversibly decreased firing frequency (from3.9 ± 1.5 Hz to 0.5 ± 0.2 Hz, n = 8, P < 0.05, Fig. 2 E and F).Furthermore, the decrease in firing frequency was associatedwith significant hyperpolarization of membrane potentials (from−46.6 ± 2.0 mV to −56.0 ± 2.1 mV, n = 8, P < 0.001, Fig. 2 Eand G). Moreover, we tested the effects of NPY on activity ofMC4R PVN neurons in external solutions with low concen-trations of Ca+2 relative to Mg+2 (Ca+2/Mg+2 = 0.2) in whichthe probability of synaptic release is significantly diminished. Inloose-patch recordings under these conditions, bath applicationof NPY reversibly inhibited firing rates in PVN neurons (from8.1 ± 1.2 Hz to 3.98 ± 0.93 Hz, n = 12, P < 0.0001, Fig. S3),suggesting that the observed effects of NPY are mediatedthrough postsynaptic mechanisms.

Effects of Leptin on MC4R PVN Neurons. Because fasting is known tobe associated with increases and decreases in the release of NPYand α-MSH, respectively, from ARC, the increases in the firingfrequency of MC4R PVN neurons observed in fasted mice cannotbe explained by virtue of ARC neuropeptide inputs only (Fig. 1A).We thus tested whetherMC4RPVNneurons can directly respondto leptin. Effects of bath applications of 35–50 nM leptin on firingactivity of MC4R PVN neurons were examined with loose-patchandwhole cell recordings (Fig. 3). Leptin inhibitedfiring activity of14 neurons and increased firing activity in 2 other neurons. In-terestingly, both neurons that were excited by leptin applicationwere located in the anterior PVN (−0.58 to −0.70 mm frombregma), whereas all neurons located in the mid- to posterior partof the PVN (−0.70 to −1.20 mm from bregma; ref. 29) wereinhibited by leptin. In work published elsewhere, we have dem-onstrated that >90% of anterior thyrotropin-releasing hormone(TRH)-positive PVN neurons are activated by both leptin andα-MSH (30).In contrast to the response in MC4R neurons of the anterior

PVN, MC4R neurons of the mid- to posterior PVN respondeduniformly to leptin application with an inhibition of action-po-tential firing (from 2.2± 0.3Hz to 1.6± 0.3Hz, n=14, P< 0.0001,Fig. 3 A and B). Because most (80–93%) of the MC4R neuronsrecorded from various metabolic states (Fig. 1) were located frommid- to posterior PVN, these neurons were binned separately forfurther analyses. In a separate set of experiments using whole cellrecording, leptin decreased firing activity of the mid- to posterior

PVN neurons (from 2.4 ± 0.2 Hz to 1.1 ± 0.2 Hz, P < 0.0001, n=17). This decrease in firing activity was associated with hyperpo-larization ofmembrane potential (from−46.8± 2.0mV to−55.8±2.0 mV, n= 17, P < 0.0005, Fig. 3 C–E). Next, we tested whetherthe observed inhibitory effects of leptin result frompostsynaptic orpresynaptic actions by examining the effect of leptin on posteriorPVN MC4R neurons while blocking ionotropic glutamate andGABA(A) receptors with PTX (200 μM) and KYN (1 mM) inwhole-cell recording. Application of 35–50 nM leptin under theseconditions resulted in a decrease in spontaneous firing frequency(from 2.5 ± 0.2 Hz to 1.1 ± 0.19 Hz, P < 0.001, n = 9), which wasassociated with hyperpolarization of membrane potentials (from−46.5± 1.7mV to−55.9mV,P< 0.05, n=9, Fig. 3F–H).We thenrepeated this experiment in some midposterior PVN MC4Rneurons in the presence of tetrodotoxin to block all activity-de-pendent neurotransmitter release. Again, leptin caused a hyper-polarization under these conditions (from −47 ± 1.2 to −57 ± 1.4

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Fig. 3. In vitro effects of leptin on firing activity of MC4R neurons in the mid-to posterior and anterior PVN. (A) Frequency histogram of effects of bathapplication of 50 nM leptin on firing activity of a MC4R PVN neuron recordedby loose-patch technique. (B) Average ± SEM of this effect obtained from 14neurons recorded from mid- to posterior PVN (n = 14, *P < 0.0001). (C) Awhole-cell recording indicates effects of 50 nM leptin on firing activity andmembrane potentials of a spontaneously firing MC4R PVN neuron. (D and E)Average ± SEM of effects of bath application of 35–50 nM leptin on firingfrequency (*P < 0.0001) and membrane potentials (*P < 0.0005) of 17 mid- toposterior MC4R PVN neurons. (F) A whole-cell recording indicates effects of50 nM leptin on firing activity and membrane potential of a MC4R PVNneuron pretreated with 200 μM PTX and 1 mM KYN. (G and H) Average ±SEM of effects of 35–50 nM bath applied leptin on firing frequency (*P <0.001) and membrane potential (*P < 0.05) of nine mid- to posterior MC4RPVN neurons pretreated with 200 μM PTX and 1 mM KYN. (I–L) Leptin acti-vates action-potential firing activity of MC4R neurons in anterior PVN. (I) Awhole-cell recording from a spontaneously firing MC4R neuron indicates thatbath application of 35 nM leptin induces an increase in firing activity associ-ated with depolarization of membrane potential. (J) The frequency histogramof this effect. (K and L) The bar graphs indicate average ± SEM of effects of35–50 nM leptin on action-potential firing frequency (K) and membranepotential (L) of seven neurons tested (in both K and L, *P < 0.005).

Ghamari-Langroudi et al. PNAS | January 4, 2011 | vol. 108 | no. 1 | 357

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mV, P < 0.05, n = 5, Fig. 3H). Thus, leptin hyperpolarized andinhibited spontaneous firing in mid- to posterior PVN MC4Rneurons by a postsynaptic mechanism.However, in MC4R neurons from anterior PVN, leptin con-

sistently increased the spontaneous firing rate of these neurons(from 1.2 ± 0.3 Hz to 2.8 ± 0.5 Hz, n = 7, P < 0.005). This in-crease in firing frequency was associated with depolarization ofmembrane potential (from −47.0 ± 1.0 mV to −41.2 ± 1.2 mV,n= 7, P < 0.005, Fig. 3 I–L). These data further indicate that theleptin-induced responses of neurons in the anterior PVN differfrom those in mid- to posterior part of the nucleus, consistentwith in our study of TRH-expressing PVN neurons (30). Wetested the hypothesis that the discrepancy in neuronal responseto leptin and α-MSH in midposterior MC4R PVN neurons maybe related to their functional role by investigating their neuro-anatomical and neurochemical properties. We thus sought toidentify MC4R PVN neurons involved in regulation of auto-nomic function by examining their ability to take up the retro-grade dye, fluorogold, injected unilaterally into the medialnucleus tractus solitarius and the T2 level of the medial spinalcord of MC4R-GFP mice. Similarly, we used systemic adminis-tration of fluorogold to label median eminence–projecting neu-rons (31). In both cases, the neurochemical identity of PVNMC4neurons as positive for TRH, corticotropin-releasing factor(CRF), or oxytocin/vasopressin was also determined. These datasuggest that the MC4R neurons in the mid- to posterior PVNcharacterized in this study are primarily brainstem-projectingneurons that coexpress oxytocin/vasopressin and CRF, whereasMC4R neurons in the anterior PVN are primarily median emi-nence–projecting neurons that coexpress TRH and CRF (see SIText, Fig. S4, and Table S1).

Circulating Leptin Increases Responsiveness of MC4R PVN Neurons toα-MSH. Previous studies have shown that leptin increases re-sponsiveness to intracerebroventricular administration of MC4Ragonists, as measured by reduction of food intake (32, 33).Furthermore, leptin can modulate the density of NPY-mediatedcurrent responses in ARC neurons (34). We therefore in-vestigated whether levels of leptin in vivo can affect re-sponsiveness of MC4R-expressing neurons to α-MSH. We thuscompared the magnitude of responses of MC4R PVN neurons toapplications of α-MSH in hypothalamic slices obtained from 16-hfasted mice injected i.p. with either leptin or saline 3 h beforedecapitation. Repeating the protocol in Fig. 1B, basal firing ac-tivity of PVN neurons from fasted animals was significantly lowerwhen mice were injected with leptin compared with saline.Furthermore, bath application of α-MSH induced excitatoryresponses in these neurons that were significantly different inposterior PVN MC4R neurons from fasted mice injected withleptin compared with those treated with saline (one-wayANOVA, P < 0.0005, Fig. 4A). Furthermore, when we comparedthe magnitude of the α-MSH–induced excitatory responses,neurons that were pretreated with leptin displayed a significantlygreater response (∼5.6-fold increase, n = 17) compared withthose with saline (∼1.5-fold increase, n = 15, Fig. 4B). Theseresults suggest that in vivo exposure to leptin modifies the re-sponsiveness of MC4R PVN neurons to α-MSH.

Leptin Increases Expression of MC4R mRNA. We next tested the hy-pothesis that leptin might increase α-MSH responsiveness by in-creasing MC4R with quantitative real-time PCR of hypothalamicMC4RmRNA in 20-h fastedmice 5 h after i.p. injection with salineor 3 μg/g of body weight of leptin. Results indicate that i.p. admin-istration of leptin significantly increased (∼24%) expression ofMC4RmRNAcomparedwith the saline-injected group (n=19–20mice, P < 0.01, Fig. 4C). These findings support the hypothesisthat leptin increases the responsiveness of PVN neurons to α-MSHby increasing the expression of the MC4R gene and possibly in-creasing receptor density on neurons (35, 36). To test the impact ofMC4R mRNA levels on energy homeostasis, we examined levelsofMC4R expression in hypothalami ofMC4R−/− (KO), MC4R +/−

(HET), and MC4R +/+ (WT) age-matched male mice fed with

normal chow (1). Our results indicate that the levels of MC4RmRNA,normalized to that ofβ-actin, inHETmice (0.57± 0.09,n=5) were significantly lower that than inWTmice (1.01± 0.04, n=12)and higher than those in KO mice (0.03 ± 0.01, n = 5, one-wayANOVA, P< 0.0001). Our data further indicate that the averages ofbody weight of HETmice (31.0 ± 0.7) were significantly higher thanWT (26.6 ± 1.0) and lower than KO mice (42.1 ± 1.0, one-wayANOVA, P< 0.0001, Fig. 4D andE), demonstrating a sharp inversecorrelation between levels of MC4R mRNA and body weight.

DiscussionAnalysis of action-potential firing frequency indicates that fastingactivates MC4R neurons in the mid- to posterior PVN. Leptinreplacement (3 h) in fasted mice reversed the fasting-inducedincrease in firing activity, suggesting that the increased firingfrequency is caused by the reduction in serum leptin levels.These data imply that midposterior MC4R PVN neurons areunder tonic inhibition by leptin. The absence of an increase infiring frequency of MC4R PVN neurons upon fasting in the ob/ob mouse further supports this hypothesis.A commonly described model for leptin action in the regula-

tion of PVN neurons invokes leptin-mediated augmentation ofproduction and release of α-MSH as well as inhibition of releaseof NPY, AgRP, and GABA after leptin action on ARC POMCand NPY/AgRP neurons, respectively (13). Previously, we haddemonstrated that α-MSH acts presynaptically to activateGABAergic inputs innervating medial parvocellular PVN neu-rons (37). Although presynaptic activity was also observed in thisstudy, we report here a dominant and direct postsynaptic effectof α-MSH on MC4R PVN neurons, increasing the firing activityof these cells. Furthermore, NPY potently and reversiblyinhibited neuronal firing activity of MC4R PVN neurons asso-ciated with hyperpolarization of membrane potentials. Thus, thetonic inhibition of firing frequency mediated by serum leptincannot be explained by leptin-mediated increase in α-MSH and/or suppression of NPY release because we show that α-MSHstimulates and NPY potently inhibits firing frequency of thesecells. AgRP was also found to potently inhibit the firing of MC4RPVN neurons (Fig. 2 C and D). This finding suggests that ourpreparation retains endogenous α-MSH release from projectionsonto the cells and/or that the MC4R retains constitutive activityin the preparation (38). These data suggest that the basal firingactivity of energy homeostasis circuits is regulated not only byleptin but also in part by the constitutive activity of the MC4R.Our results also indicate that leptin caused significant reversible

inhibition of firing activity associated with hyperpolarization ofmembrane potential in all MC4R neurons recorded in mid- toposterior PVN (Fig. 4 A–E) and that these inhibitory effects weremediated through postsynaptic mechanisms. These findings, inaddition to the lack of fasting-induced increase in firing activity ofPVN neurons observed in leptin-deficient mice, suggest that leptindirectly acts on PVN neurons to modulate their activity. Theseresults clearly require a high-affinity leptin binding site becauseexperiments were typically performed using doses as low as 50 nMleptin. The actions of leptin observed in vivo and in vitro, alongwith the observed effects of arcuate peptides, are consistent witha direct action of circulating leptin on MC4R PVN neurons, incontrast to the prevailing view of the dominance of the ARC andVMH inputs. Deletion of leptin receptors from POMC and VMHneurons, jointly, still does not cause the magnitude of obesity seenin the ob/ob mice (18). Of course, the leptin receptor acts in manyother brain regions, including additional PVN-projecting sites suchas the dorsomedial nucleus and lateral nucleus (9, 39–41). Thus,although we report here that nearly all midposterior MC4R PVNneurons are under tonic inhibition by leptin and exhibit an in-hibitory postsynaptic response to leptin, the overall regulation ofthese neurons in vivo, of course, may involve the integration ofdirect leptin actions on PVN with leptin-regulated inputs frommultiple additional nuclei other than ARC.More than 90% of TRH-expressing neurons in the anterior

PVN also exhibit a direct postsynaptic depolarizing response toboth leptin and α-MSH (30). This study also suggests that direct

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leptin action at TRH MC4R neurons in PVN may be requiredfor leptin replacement to maximally restore serum T4 levelsduring a fast. More generally, hypothalamo-pituitary discon-nection in the sheep, which ablates the ARC, suggests that theARC is required for excitation of neurons in the lateral nucleusbut not in the PVN, VMH, or dorsomedial nucleus after i.v.leptin administration (42). Thus, MC4R PVN neurons, regard-less of location or function, appear capable of responding toleptin in the absence of the ARC (Fig. S5).Our in vivo data indicated that MC4R PVN neurons activated

by fasting were located mostly (80–92%) in the mid- to posteriorregion of the nucleus. In vitro results recapitulated that finding byshowing that neurons in this region were inhibited by bath appli-cation of leptin through postsynaptic mechanisms. Revealed bytracing techniques, a significant fraction of MC4R neurons in thisregion project to the hindbrain, coexpress oxytocin/vasopressinand/or CRF, and may mediate anorexic signals (Fig. S4). Previousstudies have demonstrated a role of these peptides in relayingsatiety signals from the PVN to the brainstem (43, 44). In fact,based on characterization of electrophysiological features repor-ted for rat PVN neurons (25), our results indicate that ∼56% ofthese neurons are putative nonneuroendocrine brainstem-pro-jecting cells involved in satiety and autonomic control (Table S2).In contrast,MC4Rneurons activated by leptinweremostly locatedin the anterior PVN. These neurons are activated in fed states andinhibited during fasting states, properties consistent with up-reg-ulation of the hypothalamic–pituitary–thyroid axis by fasting.Consistentwith thismodel, our tracing and immunohistochemistrystudies performed on TRH-cremice (Table S1) indicated that thispopulation of anterior MC4R neurons project to the medianeminence and coexpress TRH (30).

In agreement with previous studies that circulating leptinincreases the anorexigenic activity of MTII (32, 33), our resultssuggest that fasting in fact decreases the responsiveness of MC4RPVN neurons to applications of α-MSH. Our results indicate thatfasting decreases hypothalamic expression of MC4R mRNA ina leptin-dependent manner. Furthermore, these findings provideevidence for plasticity of melanocortin signaling as a function ofenergy state. In parallel, there is precedent for a role of leptin inmodulating the density of NPY-mediated membrane current inARC neurons (34). Indeed, we determined that the quantity ofexpression of MC4R mRNA has a rough correlation with theseverity of obesity in that haploinsufficiency of the MC4R, whichcauses an intermediate degree of obesity relative to wild-type andknockout mice, reduces mRNA expression by ∼50%.In conclusion, this study provides a detailed analysis of the

regulation of MC4R PVN neurons likely to be involved in feedingbehavior. These data suggest that ARC to PVN circuits involved inenergy homeostasis are directly and tonically controlled by leptinin a multinodal fashion. Additionally, we identify three regulatorymechanisms important for the control of these PVN-MC4 neu-rons: (i) direct postsynaptic modulation by leptin, (ii) regulation ofMC4R mRNA expression and α-MSH responsiveness by leptin,and (iii) regulation by the constitutive activity of the MC4R sig-naling. The observation that anorexigenic α-MSH and leptin act inopposing direction to stimulate and inhibit midposterior MC4RPVN neurons, respectively, contradicts the commonly acceptedmodel of regulation of the PVN by leptin, which argues for controlvia leptin action on a homogeneous population of PVN-projectingPOMC and NPY/AgRP ARC neurons. Recent data, however,demonstrate that POMC neurons, for example, are quite hetero-geneous both neurochemically and in terms of leptin respon-siveness (45, 46), and the PVN-projecting subset of POMC neu-rons remain to be characterized.

Materials and MethodsAnimals and Housing. In all electrophysiological experiments, 26- to 60-d-oldMC4R-Tau-Sapphire transgenic (MC4R-GFP) mice and leptin-deficient ob/obmice on a C57BL/6J background were used (20). Animal husbandry is de-scribed in SI Materials and Methods. All animal experiments were approvedby the University Animal Care and Use Committee.

Electrophysiology. MC4R-GFP mice were deeply anesthetized with isofluranebefore decapitation. The brain was entirely removed and immediately sub-merged in ice-cold, gassed (95% O2 and 5% CO2) artificial cerebrospinal fluidcontaining (in mM): 126.2 NaCl, 3.1 KCl, 2 Ca Cl2, 1 Mg Cl2, 1 NaH2PO4, 26.2NaHCO3, 10 glucose, and 16.2 sucrose (pH 7.39, when gassed with 95% O2

and 5% CO2 at room temperature). Cell recordings were performed by usingpatch pipettes of 2.4-MΩ to 5-MΩ resistance when filled with a solutioncontaining (in mM): 125 K gluconate, 8 KCl, 5 MgCl2, 10 Hepes, 5 NaOH, 4Na2ATP, 0.4 Na3GTP, 15.4 sucrose, and 7 KOH, which resulted in a pH ∼7.23and osmolarity of 295–300 mosM/kg. Preparation and use of drugs is de-scribed in SI Materials and Methods.

Quantitative Real-Time-PCR. Total RNA was extracted from hypothalamictissue with the RNeasy mini kit (Qiagen) according to the manufacturer’sinstruction, and gene expression analysis was performed in 96-well platesusing TaqMan universal PCR master mix (Applied Biosystems) in a StratageneMx3000p. Details are provided in SI Materials and Methods.

Immunohistochemistry. MC4R-GFP mice were anesthetized with 2% Avertinand were injected with 200 nl of 2% fluorogold into the nucleus tractussolitarius and spinal cord at T2. Mice were allowed to recover for 1 wk andthen were injected with 20 μg of colchicine i.c.v. in 1 μL of sterile distilledwater. After 24 h, mice were then deeply anesthetized and underwent tissuefixation via transcardial perfusion with 0.9% saline followed by ice-coldfixative (4% paraformaldehyde in 0.01 M PBS). Immunohistochemistry wasthen performed as described in SI Materials and Methods.

Statistical Analysis. All data are presented as average ± SEM, and statisticalsignificance was determined by using paired t test, except where indicated,with P = 0.05 as the threshold for statistical significance.

A B

C D E

Fig. 4. Fasting reduces responsiveness of MC4R PVN neurons to bath ap-plication of α-MSH in vitro. (A) Effect of bath applications of 250 nM α-MSHon firing frequency of MC4R PVN neurons obtained from 16-h fasted miceinjected 3 h before decapitation with either 3 mg/kg leptin or saline (one-wayANOVA, *P = 0.0002). (B) Average ± SEM of magnitude of α-MSH–inducedresponse indicates that this response is greater in neurons from mice treatedwith leptin (∼5.6-fold increase, n = 17) than those treated with saline (∼1.5-fold increase, n = 15, *P < 0.0005, one-way ANOVA). (C) Hypothalamic ex-pression of MC4R gene normalized to β-actin gene obtained from fastedmice (20 h) that were injected i.p. 5 h before decapitation with either saline(n = 20) or leptin (n = 19, *P < 0.01). Data were obtained using quantitativereal-time PCR. (D) Hypothalamic expression of MC4R normalized to β-actingene obtained from MC4R−/− (KO, n = 5), MC4R−/+ (HET, n = 5), and MC4R+/+

(WT, n = 12) adult male mice fed with normal chow. Asterisk indicates allgroups are significantly different (P < 0.0001, one-way ANOVA). Data wereobtained using quantitative real-time PCR. (E) Average ± SEM of bodyweight of corresponding groups of mice as in D (*P < 0.0001, one-wayANOVA).

Ghamari-Langroudi et al. PNAS | January 4, 2011 | vol. 108 | no. 1 | 359

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ACKNOWLEDGMENTS. We thank Dr. J. Friedman (Rockefeller Uni-versity, New York) for providing MC4R-Tau-Sapphire transgenic mice,A. Hollenberg (Beth Israel Deaconess Medical Center, Boston) for TRH-cre mice, and Dr. H. Gainer (National Institutes of Health, Bethesda, MD)

for providing us with a monoclonal anti-neurophysin antibody. This workwas supported by National Institutes of Health Grant DK070332 (to R.D.C.)and Canadian Institutes of Health Research Fellowship Award 129207(to M.G.-L.).

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