Identification of neuronal subpopulations that projectfrom hypothalamus to both liver and adiposetissue polysynapticallySarah Stanleya,1, Shirly Pintoa,b,1, Jeremy Segala,c, Cristian A. Péreza, Agnes Vialea,d, Jeff DeFalcoa,e, XiaoLi Caia,Lora K. Heislerf, and Jeffrey M. Friedmana,g,2

aLaboratory of Molecular Genetics, Rockefeller University, New York, NY 10065; bMerck Research Laboratories, Rahway, NJ 07065; cDepartment of Pathology,Weill Cornell Medical College, New York, NY 10065; dGenomics Core Laboratory, Memorial Sloan Kettering Hospital, New York, NY 10065; eRenovis, SanFransisco, CA 94080; fDepartment of Pharmacology, University of Cambridge, Cambridge, CB2 1PD United Kingdom; and gHoward Hughes Medical Institute,Rockefeller University, New York, NY 10065

Contributed by Jeffrey M. Friedman, March 4, 2010 (sent for review January 6, 2010)

The autonomic nervous system regulates fuel availability andenergy storage in the liver, adipose tissue, and other organs; how-ever, the molecular components of this neural circuit are poorlyunderstood. We sought to identify neural populations that projectfrom the CNS indirectly through multisynaptic pathways to liverand epididymal white fat in mice using pseudorabies virus strainsexpressing different reporters together with BAC transgenesis andimmunohistochemistry. Neurons common to both circuits wereidentified in subpopulations of the paraventricular nucleus of thehypothalamus (PVH) by double labeling with markers expressed inviruses injected in both sites. The lateral hypothalamus and arcuatenucleus of the hypothalamus and brainstem regions (nucleus of thesolitary tract and A5 region) also project to both tissues but arelabeled at later times. Connections from these same sites to the PVHwere evident after direct injection of virus into the PVH, suggestingthat these regions lie upstreamof the PVH in a commonpathway toliver and adipose tissue (two metabolically active organs). Thesecommon populations of brainstem and hypothalamic neuronsexpress neuropeptide Y and proopiomelanocortin in the arcuatenucleus, melanin-concentrating hormone, and orexin in the lateralhypothalamus and in the corticotrophin-releasing hormone andoxytocin in the PVH. The delineation of this circuitry will facilitate afunctional analysis of the possible role of these potential command-like neurons to modulate autonomic outflow and coordinate met-abolic responses in liver and adipose tissue.

autonomic nervous system | neuronal tracing | pseudorabies virus

The autonomic nervous system plays a prominent role inmodulating carbohydrate and lipid metabolism. The original

theories of sympathetic activation (1) proposed a body-wideincrease in sympathetic outflow. However, later studies suggesteddifferential sympathetic activation on specific organs throughdistinct autonomic projections, which permits more finely tunedcontrol of metabolism (2, 3).The liver and adipose tissue play important roles in fuel storage

and release. These organs respond to altered energy availabilitywith a set of homeostatic responses mediated by humoral factorsand autonomic outflow. For example, activation of hepatic sym-pathetic aminergic and peptidergic innervation increases glucoseoutput and modulates fatty acid transport. Conversely, para-sympathetic activity decreases hepatic glucoseoutput and increasescarbohydrate storage (4). Likewise, the sympathetic innervation ofwhite adipose tissue induces lipolysis (5) and alters glucose uptake.Thus, in general, parasympathetic activity favors fuel storage,whereas sympathetic activity increases the fuel available for im-mediate use.Many CNS regions regulate autonomic outflow to hepatic or

adipose tissue to influence peripheral energy homeostasis. Inparticular, the ventromedial hypothalamus (VMH), dorsomedialhypothalamus (DMH), lateral hypothalamus (LH), and nucleus

of the solitary tract (NTS) (6) all modulate metabolic activity inliver and white fat. Neuronal tracing studies confirm these areas,and other studies innervate peripheral organs involved in car-bohydrate and lipid metabolism (7–9). However, little is knownof the site, organization, or connectivity of the CNS neural pop-ulations that effect metabolic responses.To further delineate the neural networks integrating metab-

olism in hepatic and adipose tissue, we sought to map the syn-aptically linked multineuronal efferent pathways from the CNSto liver and epididymal white fat (eWAT). We employed theretrograde neuronal tracer pseudorabies virus (PRV) to identifyCNS regions innervating liver or eWAT, and we showed sig-nificant overlap in specific molecularly defined neuronal pop-ulations that project indirectly to both organs. These data suggestthat specific hypothalamic and brainstem neural populationscontain potential command-like neurons that form a circuit tointegrate hepatic and epididymal adipose sympathetic outflow.This is in contrast to both the classic theory of sympatheticactivation as a single unit, described by Cannon (1), and themore recent data proposing independent autonomic inputs toindividual organs, which suggests coordinated sympathetic acti-vation of functionally related organs.

ResultsTime Course of Multisynaptic CNS Projections to Hepatic Tissue andWhite Adipose Tissue. We first mapped the synaptically linkedmultineuronal efferent pathways from the CNS to liver andeWAT. We established the hierarchy of the projections by fol-lowing the sites of PRV infection over time after injection ofPRV152 into the liver or eWAT. The infection course was grou-ped into five phases based on the pattern observed at differenttimes postinfection; these phases trace the course of infectionalong chains of synaptically connected neurons.Liver. At the earliest time point after hepatic PRV152 infection(3–4 days postinjection or phase I), GFP was detected in thespinal cord only. At 4–5 days postinjection (phase II), GFP wasdetected in the brainstem [motor nucleus of the vagus (10N) andpontine reticular nucleus] with limited expression in the para-ventricular nucleus of the hypothalamus (PVH), zona incerta,and parasubthalamic nucleus (SI Methods and Table S1). Areas

Author contributions: S.S., S.P., J.S., and J.M.F. designed research; S.S., S.P., J.S., A.V., J.D.,X.C., and L.K.H. performed research; C.A.P. contributed new reagents/analytic tools; S.S.,S.P., and J.S. analyzed data; and S.S., S.P., J.S., L.K.H., and J.M.F. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.1S.S. and S.P. contributed equally to this work.2To whom correspondence should be addressed. E-mail: friedj@rockefeller.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/1002790107/DCSupplemental.

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with GFP staining during phase III (5–6 days postinjection)included the NTS, intermediate and medullary reticular nuclei,dorsal periolivary region, and raphe magnus. Phase IV (6–7 dayspostinjection) revealed GFP expression in the majority ofhypothalamic sites such as DMH, VMH, arcuate nucleus of thehypothalamus (ARC), and suprachiasmatic nucleus as well ascentral amygdaloid nucleus, whereas M1 cortical infectionoccurred only at the latest stage infections (phase V; 7–8 dayspostinjection). The predominant sites of infection appearing ateach stage are illustrated in Fig. 1. Neurons in the parvocellularPVH, particularly in the posterior part (PaPo), are infected,which is in keeping with known projections to the spinal cord andbrainstem regions.White adipose tissue. Phase V PRV infection after eWAT injectionshowed a qualitatively similar pattern to that after liver injection(Table S2). Also, the time course of infection after eWAT injec-tion showed early involvement of the PVH (SI Methods and TableS2). However, unlike the hierarchy of infection after liver PRVinjection, eWAT injection resulted in early involvement of LHand cortical regions.These data indicated that the central multisynaptic outflow

pathway to liver comprises many brainstem and forebrain regionswith proximal involvement of the NTS, 10N, and PVH. Thesimilarity in the efferent pathways to liver and those to eWATled us to examine if the outflow circuits to liver and eWAT mightcontain common neurons.

Neuronal Populations in Outflow Tracts Common to Liver and AdiposeTissue. To establish the overlap in CNS sites and individual neu-rons projecting through multisynaptic pathways to liver andeWAT, we simultaneously injected PRV152 (GFP-expressing)into adipose tissue and PRV614 [red fluorescent protein (RFP)-expressing] into the liver. Neurons projecting to both tissues wouldappear yellow (i.e., red plus green). Despite the number of regionscommon to liver and eWAT infection, most neurons within theseregions were single-labeled. However, subpopulations of neuronsexpressing bothGFP andRFPwere observed in the hypothalamus(PVH, LH, and ARC) and brainstem (NTS, A5, LC, and Gi) (SIMethodsand Table S3 and Fig. S1). The sites with the greatestnumber of dual-labeled neurons were the PVH, NTS, A5 region,

LH, and ARC (Fig. 2 A–E, respectively). The time course of thedual infection (Table S3) revealed that the PVHwas the first CNSsite in which dual-labeled neurons could be identified.In summary, simultaneous injection of liver and eWAT with

isogenic PRV strains expressing different reporters identifieddual-labeled neurons in the hypothalamic PVH followed at latertimes by additional areas, predominantly the LH, ARC, NTS,and A5 region. Therefore, common neurons are involved in theoutflow circuits to both liver and eWAT.

Characterization of Common Neuronal Populations. We next soughtto chemically define the common neural populations innervatingboth liver and eWAT in areas with significant numbers of dual-labeled neurons: PVH, LH, ARC, and NTS.Common neuronal populations in the PVH. In the PVH, PRV152infection after eWAT injection colocalized with neurons expressingAVP, oxytocin (OT), corticotrophin-releasing hormone (CRF),and pro-TRH immunoreactivity. However, PRVBaBlu infectionafter liver injection colocalized with neurons expressing OT andCRFbutnotAVPorpro-TRHimmunoreactivity (Fig. 3A–F).DualPRVinjection (PRVBaBlu into liver andPRV152 intoeWAT)withsubsequent immunohistochemistry (IHC) for OT or CRH identi-fied triple-labeled neurons within the PVH. Thus, at least twounique PVH neuronal subpopulations expressing either OT orCRH send projections to both adipose tissue and liver (Fig. 3 C–Fand Fig. S2).Common neuronal populations in the LH. To characterize dual-labeledneurons in the LH, ARC, and NTS, we used BAC transgenicmouse lines expressing GFP in specific neural populations: neu-ropeptide Y (NPY), proopiomelanocortin in the arcuate nucleus(POMC), and orexin (ORX) (10–12). Mice expressing GFP inmelanin-concentrating hormone (MCH) neurons, a 17 amino acidneuropeptide that regulates food intake and body weight (13),have not been previously reported and therefore, a MCH–GFP-modified BAC was generated by homologous recombination

Spinal cord Dorsal motor nucleus of vagus

Paraventricular hypothalamic nucleus

Parasubthalamic nucleus

Nucleus of the solitary tract

Area postrema

Lateral hypothalamus

Cortex

Central nucleus of the amygdala

A5 catecholaminergic

I II III IV V

Ventral tegmental area

Fig. 1. Major sites of PRV152 infection after injection into the liver. C57Bl6mice were killed 3–8 days after injection of 1.7 × 106 pfu of PRV152 into theleft lobe of the liver (n = 3 per day for days 3–7; n = 7 for day 8). Viralinfection was visualized by immunofluorescence against GFP. The columnsshow representative areas of the five phases of infection described in TableS1. Phase I infection shows GFP expression in the spinal cord. Phase IIinfections include GFP expression in the brainstem dorsal motor 10N, PVH,and parasubthalamic nucleus (PSTh). Phase III infections include GFP immu-nofluorescence in the NTS, LH, and ventral tegmental area (VTA). Phase IVinfections include GFP expression in the area postrema (AP) and A5 regionand the central nucleus of the amygdala (Ce). Phase V infection shows GFPimmunofluorescence in the M1 region of the cortex. (Scale bar, 200 μm.)

PVH

NTS

A5

LH

ARC

A

B

C

D

E

F PVH NTS A5 LH ARC

PRV +ve neurons (eWAT) +++++ +++++ +++ +++++ +++++

PRV +ve neurons (liver) +++++ +++++ ++ +++++ +++++

% PRV eWAT infected neurons infected by PRV (liver)

5.7±2.9 14.6±6.6 13.9±6.7 16.2±3.9 9.6±1.1

% PRV (liver) infected neurons infected by PRV (eWAT)

16.8±1.8 28.1±7.9 34.7±15.2 35.2±6.9 37.2±26.4

Fig. 2. Major neuronal populations common to the outflow tracts to liverand eWAT. PRV infection of C57Bl6 mice after dual injection with GFP-expressing PRV152 into eWAT and RFP-expressing PRV614 into liver. Dual-labeled neurons, common to both liver and eWAT innervation, are presentinitially in the PVH followed by the NTS, A5 region, LH, and ARC. (A) Viralinfection within the PVH. (B) Viral infection within the NTS. (C) Viral infec-tion within the A5 region of the brainstem. (D) Viral infection within the LH.(E) Viral infection within the ARC. (Scale bar, 200 μm for left panels and50 μm for right panels.) (F) Number and average percentage ± SEM ofcoexpression for PRV-infected neuronal populations after eWAT and liverinjection. Number of positive neurons per region: +, 1–10; ++, 11–20; +++,21–30; ++++, 31–50; +++++, >50 (n = 4–5 for each region).

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(SI Methods). In the resulting BAC transgenic mice, GFP immu-noreactive neurons located in the LH and zona incerta had anidentical distribution to endogenous MCH expression as assessedby dual IHC for MCH and GFP (Fig. S3 A–D).PRV614 injection into either the liver or eWAT of MCH–GFP

mice resulted in extensive infection of GFP neurons in the LH(Fig. 4A andB). Similarly, PRV614 injection into liver or eWATofORX–GFP mice revealed PRV infection of ORX–GFP neuronsin theLH(Fig. 4C andD). Thesedata suggest thatORXandMCHneurons in the LH project to both liver and adipose tissue.Common neuronal populations in the NTS. NPY- and POMC-express-ing cells are present in the NTS, and colocalization of POMCwith GFP expression here has been confirmed in POMC–GFPmice (14). We confirmed the colocalization of NPY with GFPexpression in the NTS of NPY–GFP mice. Using IHC to visu-alize GFP–immunoreactivity (IR) neurons and in situ hybrid-ization histochemistry (ISHH) to identify NPY mRNA-expressing neurons, we found that 94% ± 4% (mean ± SEM) ofGFP–IR neurons contain the NPY mRNA label and 90% ± 3%of NPY mRNA-expressing neurons contain GFP immunor-eactivity (SI Methods and Fig. S4A).Both liver and eWAT injection resulted in colocalization of

PRV infection with GFP in the NTS of NPY–GFP mice (Fig. 5 Aand B), and thus, brainstem NPY neurons contribute to liver andeWAT innervation. However, NTS POMC–GFP neuronsshowed PRV infection only after eWAT, but not liver, injection(Fig. 5C and Fig. S4 B–E).Common neuronal populations in the ARC. In the ARC, there was noevidence of PRV infection of NPY–GFP neurons after either liveroreWAT injection (Fig. 6A andC).However, arcuatePOMC–GFPneurons were infected by PRV after either liver or eWAT injection(Fig. 6 B and D and Fig. S5). This suggests that POMC neuronscontribute to indirect outputs from the arcuate to liver and eWAT.Using established BAC transgenic mice along with IHC for

known cellular markers, we have begun to define the populations

contributing to the multisynaptic innervation of both liver andeWAT. In the PVH, CRH- and OT-expressing populations areinvolved. In the LH, MCH- and ORX-expressing cells contrib-ute. In the NTS, NPY-expressing populations are involved, andin the ARC, POMC-expressing neurons contribute to the effer-ent pathways to hepatic and adipose tissue.

APRV liver/PRV eWAT/Anti-AVP PRV liver/PRV eWAT/Anti-TRH PRV liver/PRV eWAT/Anti-CRH

PRV liver/PRV eWAT/Anti-oxytocin

B C D

E FG AVP TRH CRH OT

PRV +ve neurons (liver) ++++ +++ ++ ++++ % peptide neurons expressing PRV (eWAT) % PRV (eWAT) neurons expressing peptide% peptide neurons expressing PRV (liver)% PRV (liver) neurons expressing peptide

Peptide +ve neurons +++++ +++++ +++++ +++++

PRV +ve neurons (eWAT) ++++ +++ ++ +++

6.9 ±4.8 10.2±5.1 3.6±1.1 9.4±0.3

12.1±2.9 3.9±1.9 25.5±6.5 17±1.6

0 0 9.0±1.1 9.5±1.9

0 0 6.8±2.3 6.3±3.4

Fig. 3. Characterization of early PVH neuronal populations infected after PRV injection into eWAT and liver. Dual-labeled neurons, common to liver andeWAT innervation, express OT and CRH. Mice were killed 4–5 days after administration of PRV152 into eWAT and PRVBaBlu into liver, the earliest point atwhich infected neurons were detected in the PVH, and IHC for PVH-expressed neuropeptides was performed. Neurons with dual PRV infection and peptideexpression appeared white. Arrows indicate triple-labeled neurons. (A) PRV eWAT infection (green), PRV liver infection (red), and AVP immunofluorescence(blue). (Scale bar, 200 μm.) (B) PRV eWAT infection (green), PRV liver infection (red), and prothyrotrophin-releasing hormone (proTRH) immunofluorescence(blue). (Scale bar, 200 μm.) (C and D) PRV eWAT infection (green), PRV liver infection (red), and CRH immunofluorescence (blue). (Scale bar, 200 μm on the leftand 50 μm on the right.) (E and F) PRV eWAT infection (green), PRV liver infection (red), and OT immunofluorescence (blue). (Scale bar, 200 μm on the left and50 μm on the right.) (G) Number and average percentage ± SEM of coexpression for each PVH neuropeptide for PRV-infected neuronal populations aftereWAT and liver injection. Number of positive neurons per region: +, 1–10; ++, 11–20; +++, 21–30; ++++, 31–50; +++++, >50 (n = 3–4 for each region).

B

C D

PRV liver/MCH PRV liver/Orexin

PRV eWAT/MCH PRV eWAT/Orexin

MCH ORX

Peptide +ve neurons +++++ +++++

PRV +ve neurons (eWAT) +++++ ++++

PRV +ve neurons (liver) +++++ ++++

% peptide neurons expressing PRV (eWAT) 10.6±0.2 4.0±0.4

% PRV (eWAT) neurons expressing peptide 2.6±0.9 11.0±1.0

% peptide neurons expressing PRV (liver) 7.7±0.9 3.9±1.4

% PRV (liver) neurons expressing peptide 4.4±1.8 14.3±4.3

A E

Fig. 4. Involvement of LH MCH and ORX neuronal populations in outputpathways to liver and eWAT. Both MCH and ORX neurons in the LH areinvolved in the outflow circuits to liver and adipose tissue. (A) Colocalizationof liver PRV infection (red) and MCH neurons (green). Merged photo-micrographs show colocalization of liver PRV infection in a subpopulation ofMCH neurons (yellow, indicated). (Scale bar, 50 μm.) (B) Colocalization ofORX neurons (green) and liver PRV infection (red). Liver viral infection andORX immunoreactivity were observed in a subpopulation of ORX neurons(yellow, indicated). (Scale bar, 50 μm.) (C) Localization of MCH neurons(green) and eWAT PRV infection (red). (Scale bar, 50 μm.) (D) Localization ofORX neurons (green) and eWAT PRV infection (red). Colocalization of eWATPRV infection and ORX immunoreactivity was observed in a subpopulationof ORX neurons. (Scale bar, 50 μm.) (E) Number and average percentage ±SEM of coexpression for each LH peptide for PRV-infected neuronal pop-ulations after eWAT and liver injection. Number of positive neurons perregion: +, 1–10; ++, 11–20; +++, 21–30; ++++, 31–50; +++++, >50 (n = 4 foreach region).

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Characterization of Neuronal Populations Innervating the PVH. ThePVH was the initial site with double-labeled neurons after dualPRV infection of liver and adipose tissue and later infection inthe LH, ARC, A5, and NTS. These later infections may haveoccurred independently of the PVH or after transsynaptic spreadfrom PVH neurons to these regions. We sought to determine ifPRV injections into the PVH recapitulated the infection in LH,

ARC, A5, and NTS neurons seen after liver or eWAT injection.Therefore, we unilaterally injected PRV614 directly into thePVH (rather than the liver or eWAT). Two days after injection,PRV infection was evident in the LH, ARC, NTS, and A5 region(SI Methods and Fig. S6 A–F) as seen after liver or eWATinjection. Similarly, PRV infection after PVH injection colo-calized with MCH–GFP and ORX–GFP neurons in the LHalong with POMC–GFP neurons in the ARC and NTS NPY–

GFP neurons. However, unlike eWAT and liver injections, therewas also infection of arcuate NPY neurons.

DiscussionIn this report, we combined PRV tracing, BAC transgenesis, andIHC to define CNS neuronal populations that project indirectlythrough multisynaptic pathways to liver and eWAT in mice.After infection of the tissues with two isogenic PRV strains, dual-labeled neurons in the PVH identified a subpopulation thatinnervates both tissues. Additional neurons in the hypothalamicLH and ARC and brainstem NTS and A5 region also project toboth tissues but are labeled at later times. These data, togetherwith data showing connections between these regions and thePVH, strongly suggest that the LH, ARC, NTS, and A5 regionsare upstream of the PVH in this pathway. In aggregate, theseregions constitute an outflow pathway to two key metabolicallyactive organs. These findings also identify discrete neuronalpopulations that may control autonomic outflow to both hepaticand adipose tissue and may orchestrate the coordinated controlof peripheral metabolism.

Efferent Outputs to Hepatic and Adipose Tissues. Our single-organPRV injections revealed that neurons in multiple brainstem andforebrain regions project to liver and eWAT, and many of thesesites are common to both innervation pathways. These data areconsistent with studies implicating the hypothalamic PVH andLH in hepatic autonomic innervation (15) and regulation ofhepatic glucose metabolism (15, 16). Our findings are also in

A B C

PRV eWAT/Anti-POMCPRV eWAT/Anti-NPYPRV liver/Anti-NPY

D NPY POMC

Peptide +ve neurons +++++ +++++

PRV +ve neurons (eWAT) ++ ++++

PRV +ve neurons (liver) ++++ ++++

% peptide neurons expressing PRV (eWAT) 2.3±1.6 7.9±3.0

% PRV (eWAT) neurons expressing peptide 4.9±1.7 2.2±0.1

% peptide neurons expressing PRV (liver) 7.9±1.8 0

% PRV (liver) neurons expressing peptide 8.6±4.1 0

Fig. 5. Involvement of NPY and POMC brainstem neuronal populations inoutput pathways to liver and eWAT. Brainstem NPY neurons contribute tothe efferent pathways to both liver and eWAT, but POMC neurons are onlyinvolved in the outflow circuit to adipose tissue. (A) Colocalization of NPY(green) and PRV infection (red) in the NTS after injection of PRV614 into theliver of NPY–GFP mice. An overlay of the GFP and PRV immunofluorescenceimages indicates colocalization of GFP expression and PRV infection (yellow,marked). (Scale bar, 50 μm.) (B) Colocalization of NPY (green) and PRVinfection (red) in the NTS after injection of PRV614 into the eWAT of NPY–GFP mice. An overlay of the GFP and PRV immunofluorescence imagesindicates colocalization of GFP expression and PRV infection (yellow,marked). (Scale bar, 50 μm.) (C) Colocalization of POMC–GFP expression(green) and eWAT PRV infection (red) is observed. (Scale bar, 50 μm.) (D)Number and average percentage ± SEM of coexpression for each brainstempeptide for PRV-infected neuronal populations after eWAT and liver injec-tion are shown. Number of positive neurons per region: +, 1–10; ++, 11–20;+++, 21–30; ++++, 31–50; +++++, >50 (n = 4 for each region).

A B

C D

PRV liver/POMCPRV liver/NPY

PRV eWAT/NPY

E

PRV eWAT/POMC

NPY POMC

Peptide +ve neurons +++++ +++++

PRV +ve neurons (eWAT) ++ +++++

PRV +ve neurons (liver) +++++ +++++

% peptide neurons expressing PRV (eWAT) 0 5.6±2.5

% PRV (eWAT) neurons expressing peptide 0 6.5±2.6

% peptide neurons expressing PRV (liver) 0 2.4±1.9

% PRV (liver) neurons expressing peptide 0 13.1±9.0

Fig. 6. Involvement of ARC NPY and POMC neuronal populations in output pathways to liver and eWAT. Only ARC POMC neurons are involved in the outputpathways to both liver and eWAT with no evidence of ARC NPY neuronal involvement. No evidence of colocalization of hypothalamic NPY neurons (green)and PRV infection (red) after injection of PRV614 into (A) the liver or (B) eWAT of NPY–GFP mice. (Scale bar, 200 μm.) Colocalization of ARC POMC neurons(green) and PRV infection (red) after injection of PRV614 into (C) the liver or (D) eWAT of POMC–GFP mice. (Scale bar, 50 μm.) (E) Number and percentage ±SEM of coexpression for each ARC neuropeptide for PRV-infected neuronal populations after eWAT and liver injection. Number of positive neurons perregion: +, 1–10; ++, 11–20; +++, 21–30; ++++, 31–50; +++++, >50 (n = 4 for each region).

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keeping with prior PRV studies in other species tracing fromliver or eWAT (8, 9, 17–19).One technical point is that the proportion of dual-labeled

neurons is typically small. This could mean that only a smallfraction of neurons are elements of the outflow tracts to botheWAT and liver. However, the number of neurons common toboth outflow pathways may be an underestimate for technicalreasons. Prior studies showed marked variation in the extent ofdual infection, even for viral mixtures (PRV152 and PRVBaBlu)injected into the same organ (10–30% of cells show no evidenceof dual infection) and asymmetrical infection after simultaneousinfection of bilateral organs (20). A reason for this is a limitedwindow in which prior viral infection does not preclude coin-fection by a second viral strain (21). This factor renders dualPRV tracing most useful for qualitative rather than quantitativeassessment. Dual-infected neurons after PRV injection intodifferent tissues can only occur when PRV has retrogradelytransmitted along their neuronal pathways to a common point.In many experiments, there will be a fraction of neurons that liein a common pathway where PRV transmission from one sitearrives outside the time window in which dual infection canoccur, possibly leading to underestimation of the number ofneurons common to two neural pathways.

Command-Like Neurons. In his early theories of sympathetic acti-vation, Cannon (1) proposed that the sympathetic nervous systemresponded as a single entity with activation of all components inresponse to threat (1). Later work refuted this by describingindependent regulation of sympathetic outflow to individualorgans (2). However, Loewy and colleagues (22) identified com-mon neurons in the sympathetic outflow to both heart and adrenalthat might result in coordinated activity of both tissues whenrequired, such as in the flight-or-fight response (22). The currentdata suggest this may also be the case for metabolically activeorgans such as liver and adipose tissue. Several CNS areas, pri-marily hypothalamic PVH, LH, and ARC and brainstemNTS andA5 regions, contain common neural populations that project toliver and eWAT. These cells provide indirect efferent outputs toboth sites. This is an anatomic characteristic of command neuronsin vertebrates where neurons from multiple sites converge on asingle command neuron (22). Altered activity in these dual-labeled neurons may coordinate metabolic function in both liverand eWAT to participate in an integrated response to fuelavailability. Further studies will be required to establish thefunctional properties of these common cells, and the delineationof molecular markers for these neurons will make it possible toexplore the effects of modulating their activity (such as by usingchannel rhodopsin) (23).

PVH as Initial Site of Integration. PVH neurons were the first toshow dual labeling after infection of liver and eWAT. Theseneurons are known to project directly to the spinal sympatheticpreganglionic neurons of the intermediolateral cell column. Duallabeling in LH, ARC, NTS, and A5 occurred later, either throughsynaptic connection to the PVH or an independent route to liverand adipose tissue. PRV injection directly into the PVH resultedin LH, ARC, NTS, and A5 infection; this suggests that theseregions project to the PVH, which may then serve as a point ofintegration in the innervation pathway to liver and eWAT.Anterograde tracing studies in other species show efferent

connections between the LH and PVH (24), the ARC and PVH(25), and the NTS and PVH (26). No direct efferent connectionsfrom A5 region to the PVH have been described. Based onretrograde tracing, we report synaptic connections between theLH, ARC, NTS, and A5 regions, and the PVH also exist in mice.

Neuronal Populations Projecting to Peripheral Sites. Using BACtransgenic animals and immunohistochemical markers, we iden-

tified several neuronal populations common to hepatic and adi-pose tissue innervation. Within the PVH, dual-labeled neuralsubpopulations express CRH and OT. These populations havealso been implicated in autonomic outflow through the stellateganglion (27). In addition, both intracerebral CRHandOTmodifyautonomic activity (28, 29). In the LH, ORX and MCH cell pop-ulations project to both liver and adipose tissue. These neuralpopulations are also linked to sympathetic outflow to the adrenalgland and through the stellate ganglion (30, 31).ARC neurons infected after either liver or eWAT injection

express POMC. Central injection of melanocortin agonistsincreases sympathetic activity to WAT (3), and melanocortinreceptor 4mRNAhas been colocalized to infected cells after PRVinfection of WAT (32). Melanocortin agonists also modulatehepatic glucose metabolism. However, this study also showsPOMC neurons to be synaptically linked to eWAT and liver. Ourstudies also showdual-labeled neurons in theNTS, a region knownto modulate multiple sympathetic responses (33–35). We foundNTSNPYbut not POMCneurons innervate both liver and eWAT,and thus, they may be part of the common outflow to liver andeWAT. Finally, we identified dual-labeled neurons in the A5region, an area containing catecholaminergic neurons, that havebeen implicated in sympathetic outflow to many organs (27, 36).Our findings also confirm those from prior tracing studies fromwhite fat or liver in other species (9, 19).These data defining the dual-labeled neurons in outflow tracts

to liver and adipose tissue will enable future functional studies todetermine the role of these common neural populations inintegration of metabolic function.

Future Work to Functionally Characterize Marked Neurons. Identify-ing molecular markers, such as CRH and OT, for the neuronscommon to liver and fat innervation will now enable us to assessif these populations regulate metabolic activity in these tissues.Targeting functional cassettes, such as channel rhodopsin (23),to these cells will determine whether selective activation is suf-ficient to modulate autonomic activity to both liver and eWAT.Fluorescent labeling of the common population would allow theelectrical properties of the neurons to be examined. Addition-ally, analysis of gene expression in the common cell populations,by immunoprecipitation of tagged polysomes (37), may provideinformation about the intracellular pathways linking alterationsin metabolic conditions and sympathetic activity. Ultimately,these approaches may enable us to formally test the hypothesisthat these neurons are necessary and sufficient to modulatephysiologic functions, criteria that, if satisfied, would establishthese cells as command-like neurons.In summary, we used a combination of genetic tools to study

the CNS innervation of liver and eWAT. We used PRV Bartha-expressing reporter proteins to identify the central neural sitesinnervating liver or eWAT in mice, and we showed the consid-erable overlap in the anatomy of the outflow circuits to theseorgans. Dual-tracing studies defined neuronal subpopulationsthat innervate both hepatic and adipose tissue. Furthermore,PRV tracing in BAC transgenic mice expressing GFP in selectedneuronal populations allowed us to characterize neurons in themultisynaptic pathway to both organs. We found that subpop-ulations of neurons expressing CRH and OT in the PVH, ORXand MCH in the LH, NPY in the NTS, and POMC in the ARCcontribute to the multisynaptic pathways to both liver andeWAT. Defining these common populations will enable futurestudies to test if these neurons integrate metabolic activity inliver and eWAT. This combination of viral and transgenicstrategies may also prove fruitful in delineating and character-izing the central sites innervating many peripheral organs andfacilitate studies of the central coordination of metabolic pro-grams in anatomic disparate but functionally related organs.

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MethodsAnimals, Pseudorabies Virus Overview, Preparation, and Injection.Male C57Bl6and transgenic mice were used. Animal care and experimental procedureswere performed with approval from the Animal Care and Use Committee ofRockefeller University (protocol 06074) under established guidelines.

Three isogenic PRV–Bartha recombinants were used in the studies:PRV152, PRV614, and PRVBaBlu encoding GFP, RFP, and β-galactosidase,respectively, in the viral gG locus. Stocks of PRV152 (3.4 × 108 pfu/mL), PRVBaBlu (5 × 108 pfu/mL; L. Enquist, Princeton, NJ), and PRV614 (3 × 108 pfu/mL;B. Banfield, Ontario, Canada) were prepared as previously described (38),and aliquots were stored at −80 °C before use.

Anesthetized mice were injected with PRV (3 μL) over 30 seconds using aHamilton syringe, and the needle was left in place for 1 additional minute.Injections were made into the left lobe of the liver and/or the right epi-didymal fat pat. Injected mice were perfused at specified time points orwhen symptoms of infection appeared, usually between 6 and 8 dayspostinjection. For PRV injection into the PVH, anesthetized animals wereplaced in a stereotactic frame and 150 nL of PRV152, with 5% fluorescentbeads, was injected over 5 minutes at coordinates 0.8 mm posterior, 0.15lateral, and 5.5 mm down from bregma.

Immunofluorescence and GFP Colocalization. Immunofluorescence and doublelabeling were performed as previously described (38) (SI Methods). C57Bl6mice infected with PRV and stained for CRH or pro-TRH were treated withcolchicine to enhance cell body staining as described in SI Methods.

Single-labeled images were acquired using a Zeiss Axioplane microscopeand captured digitally with separate band-pass filters (SI Methods) using themultichannel module of the AxioVision Zeiss software. Dual- and triple-labeled sections were also imaged by confocal microscopy (LSM 510 laser-scanning confocal microscope; Carl Zeiss MicroImaging). Additional detailsare included in SI Methods. Structures were defined according to their mor-phology comparedwith sections illustrated in TheMouse Brain in StereotacticCo-ordinates (39). Nomenclature was based on Paxinos and Franklin (39).

Generation of MCH–GFP BAC Transgenic Mice. The MCH–GFP transgenic mousewas created using BAC transgenic technology as previously described (40)(SI Methods).

ISHH and IHC in NPY–GFP Mice. Coronal brain sections (25 μm) were probedwith an NPY antisense riboprobe in a modification of reported procedures(41) (SI Methods).

ACKNOWLEDGMENTS. We thank Friedman Laboratory members for helpfuldiscussions and Oliver Marston from the Heisler Laboratory for assistancewith image preparation and analysis. We thank S. Korres for assistance withpreparing the manuscript. We thank the staff of the Rockefeller UniversityBioimaging Resource Center for their technical support in confocal imaging.S.S. was supported by a Medical Research Council Clinician Scientist Fellow-ship. C.A.P. was supported by the Sjögren’s Syndrome Foundation. L.K.H.was supported by the National Institutes of Health Grant DK065171-03and the Wellcome Trust. This work was funded by the National Institutesof Health Grant DA018799-03.

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