Distinct Populations of Presympathetic- Premotor Neurons Express Orexin or Melanin ...

16 Pages·2007·4.4 MB·English

THE JOURNAL OF COMPARATIVE NEUROLOGY 505:586–601 (2007)
Distinct Populations of Presympathetic-
Premotor Neurons Express Orexin or
Melanin-Concentrating Hormone in the
Rat Lateral Hypothalamus
ILAN A. KERMAN,* RENE´ BERNARD, DEVIN ROSENTHAL, JAMES BEALS,
HUDA AKIL, AND STANLEY J. WATSON
MolecularandBehavioralNeuroscienceInstitute,UniversityofMichigan,
AnnArbor,Michigan48109
ABSTRACT
Orexin and melanin-concentrating hormone (MCH) have been implicated in mediating a
varietyofdifferentbehaviors.Theseincludesleepandwakefulness,locomotion,ingestivebehav-
iors,andfight-or-flightresponse,aswellasanxiety-andpanic-likebehaviorsinrodents.Despite
suchdiversity,alltheseprocessesrequirecoordinatedrecruitmentoftheautonomicandsomato-
motorefferents.Wehavepreviouslymappedthelocationsofpresympathetic-premotorneurons
(PSPMNs)intheratbrain.Theseputativedual-functionneuronssendtrans-synapticprojections
to somatomotor and sympathetic targets and likely participate in somatomotor-sympathetic
integration.Asignificantportionoftheseneuronsisfoundwithinthedorsomedial(DMH)and
lateral hypothalamus (LH), areas of the brain that contain MCH- and orexin- synthesizing
neurons in the central nervous system. Thus, we hypothesized that hypothalamic PSPMNs
utilizeMCHororexinastheirneurotransmitter.Totestthishypothesis,weidentifiedPSPMNs
by using recombinant strains of the pseudorabies virus (PRV) for trans-synaptic tract tracing.
PRV-152,astrainthatexpressesenhancedgreenfluorescentprotein,wasinjectedintosympa-
thectomizedgastrocnemiusmuscle,whereasPRV-BaBlu,whichexpresses(cid:1)-galactosidase,was
injectedintotheadrenalglandinthesameanimals.Byusingimmunofluorescentmethods,we
determinedwhetherco-infectedneuronsexpressMCHororexin.Ourfindingsdemonstratethat
PSPMNs synthesizing either MCH or orexin are present within LH, where they form two
separatepopulations.PSPMNslocatedaroundthefornixexpressorexin,whereasthoselocated
aroundthecerebralpedunclearemorelikelytoexpressMCH.ThesetwoclustersofPSPMNs
within LH likely play distinct functional roles in autonomic homeostasis and stress coping
mechanisms.J.Comp.Neurol.505:586–601,2007. ©2007Wiley-Liss,Inc.
Indexingterms: somatomotor-sympatheticintegration;somatomotorcontrol;centralautonomic
circuits;pseudorabiesvirus;peptides;transneuronaltracing
Orexins(orexin-Aandorexin-B;alsoknownashypocre- manakaetal.,2003;Akiyamaetal.,2004;Boutreletal.,
tin1andhypocretin2,respectively)wereinitiallydiscov- 2005; Harris et al., 2005; Narita et al., 2006; Sakurai,
ered as ligands for two orphan G-protein-coupled recep-
tors (de Lecea et al., 1998; Sakurai et al., 1998). These
peptides are derived from a common precursor molecule,
prepro-orexin,whichiscleavedtoformorexin-A(33amino Grantsponsor:thePritzkerNeuropsychiatricDisordersResearchCon-
sortium;Grantsponsor:NationalInstitutesofHealth;Grantnumber:P01
acidslong)andorexin-B(28aminoacids)(Sakuraietal.,
MH042251(toS.J.W.);Grantsponsor:theUniversityofMichiganCom-
1998;Willieetal.,2001).IntheirinitialreportSakuraiet prehensiveDepressionCenterInnovationFundGrantProgram(toI.A.K.
al. (1998) described the structure of these peptides and and R.B); Grant sponsor: The Mental Health Research Association
(NARSAD)YoungInvestigatorAward(toI.A.K.).
demonstratedthattheystimulateshort-termfoodintake.
*Correspondenceto:IlanA.Kerman,MolecularandBehavioralNeuro-
Subsequentinvestigationshaveconfirmedthatthesepep-
science Institute, 205 Zina Pitcher Place, University of Michigan, Ann
tides stimulate arousal and feeding behavior in rats and Arbor,MI48109.E-mail:[email protected]
mice(Edwardsetal.,1999;Haynesetal.,2000,2002)and Received25April2007;Revised2July2007;Accepted23August2007
have also implicated them in the regulation of emotion, DOI10.1002/cne.21511
energy homeostasis, reward, and drug addiction (Ya- PublishedonlineinWileyInterScience(www.interscience.wiley.com).
©2007WILEY-LISS,INC.
TheJournalofComparativeNeurology.DOI10.1002/cne
PSPMNSANDHYPOTHALAMICPEPTIDES 587
2007). It has also been demonstrated that the orexins MCH. To test this hypothesis we identified PSPMNs by
participateinregulatinglocomotoractivityaspartofmo- usingourpreviouslydevelopedretrogradetrans-synaptic
tivatedbehaviors,suchasforagingforfoodfollowingfast- tract-tracing paradigm and then used triple-label immu-
ing (Yamanaka et al., 2003) and that they are part of an nofluorescence to determine whether these cells synthe-
integratedcardiovascularandlocomotorresponsetoacute sizeorexinsorMCH.
stress (Zhang et al., 2006). In addition, intracerebroven-
tricular administration of orexin-A leads to increases in
meanarterialpressureandheartrateinawakebehaving MATERIALS AND METHODS
rats(Samsonetal.,2007).
All the procedures regarding animal use in this study
Orexin-synthesizingneuronsinthecentralnervoussys-
conformedtotheGuidefortheCareandUseofLaboratory
tem are found exclusively within the lateral (LH) and
AnimalsoftheNIHandwereapprovedbytheUniversity
dorsomedial (DMH) hypothalamic nuclei (Sakurai et al.,
of Michigan’s University Committee on Use and Care of
1998;Swansonetal.,2005).Withintheseregions,orexin-
Animals.
synthesizing neurons are intermingled with those neu-
rons that synthesize melanin-concentrating hormone
Antibody specificity and distribution of
(MCH). Although these two populations of hypothalamic
MCH- and orexin-synthesizing neurons
neuronsarelocatedwithinthesameareas,theyareneu-
rochemically distinct, as MCH-positive cells do not syn- In our initial experiments we sought to compare the
thesize orexins and vice versa (Bayer et al., 2002; Swan- distribution of MCH- and orexin-synthesizing neurons in
sonetal.,2005). the rat brain. Adult male Sprague-Dawley rats (n (cid:2) 2;
MCHisalsolocatedinthelateralhypothalamusandis CharlesRiver,Wilmington,MA)wereusedinthesestud-
a 19-amino-acid-long neuroactive peptide that is derived ies.Animalsweredeeplyanesthetizedwith1mli.p.of50
fromtheprecursorprepro-MCH(Bittencourtetal.,1992). mg/ml pentobarbital sodium solution and transcardially
MCH was originally discovered in fish but has subse- perfusedwith100–150mlofphysiologicalsalinefollowed
quentlybeenidentifiedinmammals,inwhomitisthought by 400–500 ml of a 4% paraformaldehyde solution con-
to regulate energy balance (Pissios et al., 2006). In addi- taining 1.4% l-lysine and 0.2% sodium meta-periodate
tion to energy balance, MCH neurotransmission has also (PLP)(McLeanandNakane,1974).Brainswerethenex-
beenimplicatedinregulatinglocomotoractivity(Marshet tracted,postfixedinPLPovernight,andimmersedin20%
al., 2002; Segal-Lieberman et al., 2003), and inactivation sucrose until they sank to the bottom of the container.
of the MCH receptor 1 leads to increased wheel running Tissue was then sectioned coronally on a freezing mic-
(Zhou et al., 2005). MCH neurotransmission is also rotome at a thickness of 40 (cid:3)m and collected into six
thought to play a role in mediating anxiety- and adjacent bins. Tissue was then stored at (cid:4)20°C in cryo-
depression-like behaviors in rodents (Borowsky et al., protectant (30% sucrose, 30% ethylene glycol, 1%
2002). Furthermore, intracerebroventricular infusions of polyvinyl-pyrrolidine [PVP-40]) until immunohistochemi-
MCH increase rapid eye movement sleep (Verret et al., calprocessingwasconducted.
2003)andproducebradycardicandhypotensiveresponses To study the distribution of MCH-positive neurons, we
in rats (Messina and Overton, 2007), suggesting that used rabbit anti-MCH serum (Phoenix Pharmaceuticals,
MCHneurotransmissionalsoparticipatesinregulationof Burlingame, CA, cat. #: H-070-47, lot #: 00606). This an-
theautonomicnervoussystemandthesleep/wakecycle. tibody was raised against full-length peptide (sequence:
Taken together, these data suggest that orexins and Asp-Phe-Asp-Met-Leu-Arg-Cys-Met-Leu-Gly-Arg-Val-
MCHparticipateincoordinatingactivitiesofsomatomotor Tyr-Arg-Pro-Cys-Trp-Gln-Val) and has been shown to
and autonomic systems and that they do so within the have 100% cross-reactivity against MCH and 0% cross-
contextofmotivatedbehaviors.Wehavepreviouslyuseda reactivity against orexins, agouti-related peptide, leptin,
virally mediated trans-synaptic tract-tracing approach to (cid:5)-melanocyte-stimulating hormone, and neuropeptide Y
map the locations of neurons that send polysynaptic pro- (manufacturer’stechnicalinformation).
jectionstoskeletalmuscleandtotheadrenalgland(Ker- To prevent nonspecific binding, tissue was first soaked
man et al., 2003, 2006a,b). We have proposed that these in blocking solution that consisted of 0.3% Triton X-100
neurons(referredtohereaspresympathetic-premotorneu- (TX-100), 1% normal goat serum (NGS), and 1% bovine
rons[PSPMNs])havedualfunctionsinregulatingactivi- serumalbumin(BSA)in0.1Mphosphatebuffer(PB;pH
ties of the sympathetic and somatomotor systems. Al- 7.4).Theantibodywasthendissolvedinthesameblocking
though PSPMNs are widely distributed throughout the mix and reacted with tissue at 1:10,000 or 1:15,000 for 1
brain, major clusters of these neurons are found within hour at room temperature followed by 48 hours at 4°C.
areas thought to mediate motivated behaviors such as The tissue was then reacted with biotinylated goat anti-
responses to stress (Kerman et al., 2003, 2006a,b). Such rabbit IgG (Vector, Burlingame, CA) at 1:200 in blocking
areasincludeLHandDMH,bothregionsthathavebeen solution for 2 hours at room temperature. Following a
implicatedinmediatingstressresponsesaswellasregu- briefrinsein0.1MPB,thetissuewasthenreactedwith
latingfoodintake(Kermanetal.,2006a).Theseareasalso the VectaStain ABC System (Vector), with A and B re-
contain all the orexin-synthesizing neurons and the ma- agents dissolved at 1:200 each in blocking solution. The
jorityofMCH-synthesizingneuronsinthecentralnervous reaction was then visualized with 0.04% diaminobenze-
system(Bittencourtetal.,1992;Sakuraietal.,1998). dine(DAB)aschromogenand2(cid:6)10(cid:4)5%H O in0.1M
2 2
Thus,giventheproposedroleofPSPMNsinregulating sodiumacetate.
physiological aspects of motivated behaviors and the To verify the specificity of this antibody further, we
known role of orexins and MCH in coordinating somato- performed a blocking study in which adjacent sections
motor and autonomic functions, we hypothesized that a were incubated with the rabbit anti-MCH serum (as de-
subset of hypothalamic PSPMNs synthesize orexins or scribedabove)andwithrabbitanti-MCHserumpreincu-
TheJournalofComparativeNeurology.DOI10.1002/cne
588 I.A.KERMANETAL.
batedwith50(cid:3)MoftheMCHpeptide(PhoenixPharma- tion. Alternatively, animals were injected i.p. with a mix
ceuticals,cat.#:070-47,lot#:424859). ofketamineandxylazine(60mg/kgketamineand7mg/kg
To study the distribution of orexin-synthesizing neu- xylazine). Once a surgical plane of anesthesia was
rons, we used an antibody raised against orexin-A. We achieved such that there was no spontaneous movement
usedrabbitpolyclonalantibody(Abcam,Cambridge,MA, andtherewerenowithdrawalresponsestotailand/orfoot
cat. #: ab6214, lot #: 38610), which was raised against a pinch,hindlimbsympathectomywasperformed.
synthetic peptide (sequence: Cys-Arg-Leu-Tyr-Glu-Leu- In addition to somatic motor efferents, the hindlimb is
Leu-His-Gly-Ala-Gly-Asn-His-Ala-Ala-Gly-Ile-Leu-Thr- also innervated by sympathetic efferents that project to
Leu),correspondingtoaminoacids14–33ofcoworexin-A, blood vessels, sweat glands, and other smooth muscle
andconjugatedtoLys-Leu-Hisbyaglutaraldehydelinker targets(Ja¨nigandMcLachlan,1992).Topreventinfection
(manufacturer’s technical information). This peptide has of sympathetic pathways following injection of PRV-152
100% amino acid identity with rat orexin-A, and it con- into the gastrocnemius muscle, the hindlimb was surgi-
tainsthreeregionsofidentitywithorexin-Bforatotalof cally sympathectomized in all animals by using a previ-
13 identical amino acid residues between orexin-A and ously described approach (Kerman et al., 2003). Briefly,
orexin-B (Sakurai et al., 1998). This antibody has been thelumbarsympatheticnervewasdissectedviaaventral
extensively tested to show that it cross-reacts to detect laparotomyandwasextirpatedfromtheleveloftherenal
bothorexin-Aandorexin-Bandthatitonlystainsorexin- artery caudal to the bifurcation of the abdominal aorta.
synthesizing neurons (Nambu et al., 1999). Tissue was Neural plexuses running along the descending aorta and
incubated with this antibody diluted to 1:5,000 and then inferiorvenacavawerealsostrippedoffundermicroscopic
processedandvisualizedasdescribedabove. observation by using fine forceps. The abdominal aorta
and the inferior vena cava were then swabbed with 10–
Overview of tract-tracing studies
20% phenol dissolved in ethylene glycol. The abdominal
MaleSprague-Dawleyrats(n(cid:2)11;CharlesRiver)were musculature was closed with sutures, and the overlying
used in tract-tracing studies. In these experiments we skin was closed with surgical staples. This procedure is
employedtransgenicrecombinantsofanattenuatedpseu- effective at removing the large majority of sympathetic
dorabiesvirus(PRV)strain,PRV-Bartha,fortransneuro- efferents to the hindlimb, as evidenced by the lack of
nal tracing of multisynaptic pathways innervating adre- infectionintheintermediolateralcellcolumninthesame
nal gland and gastrocnemius muscle. PRV has animals that exhibited robust infections of lumbar mo-
preferential tropism for axonal terminals (Vahlne et al., toneurons(Kermanetal.,2003,2006a,b).
1978, 1980). It is transported in the retrograde direction Following sympathectomy and a 2–10-day recovery pe-
fromtheterminalstothecellbodywheretheviralgenome riod, the animals were reanesthetized and injected with
isreplicatedinthenucleus(Enquistetal.,1998).Capsids PRV-152 into either their right (n (cid:2) 2) or left (n (cid:2) 9)
are assembled and filled with viral DNA in the nucleus, gastrocnemius muscle. Following a 24–32-hour recovery
acquire the mature envelope from a late Golgi compart- period, the same rats were reanesthetized and were in-
ment, and are transported to sites of afferent synaptic jected with PRV-BaBlu into their left adrenal glands.
contact,wherecell-to-cellretrogradetransneuronaltrans- Choiceofanestheticregimehadnoeffectonviralinfection
mission of infection occurs (Card et al., 1993; Enquist et ortransport.
al., 1998). The two viral recombinants that we employed Attheconclusionofthesurvivalperiodfollowingtrans-
were PRV-152 and PRV-BaBlu. Both are derived from port of viral tracers, animals were deeply anesthetized
PRV-Bartha,whichisanattenuatedformoftheparental with 1 ml i.p. of 50 mg/ml pentobarbital sodium solution
strain,PRV-Becker.PRV-BaBlucontainsthelacZgeneat and transcardially perfused with 100–150 ml of physio-
the gG locus and produces (cid:1)-galactosidase ((cid:1)-gal) under logicalsalinefollowedby400–500mlofa4%paraformal-
the control of the viral gG promoter (Kim et al., 1999). dehydesolutioncontaining1.4%l-lysineand0.2%sodium
PRV-152 carries the gene coding for enhanced green flu- meta-periodate(PLP)(McLeanandNakane,1974).
orescent protein (eGFP) at the gG locus, which is consti-
Double-virus injections
tutively expressed under control of the cytomegalovirus
immediate early promoter (Smith et al., 2000). Previous Viral recombinants used in the present studies were
studieshavedemonstratedthatPRV-152andPRV-BaBlu harvestedfrompigkidneycellculturesatatiterof108to
aretransportedtranssynapticallyinaretrogrademanner, 109 pfu/ml. Viral stocks were aliquoted in 50-(cid:3)l volumes
similarly to PRV-Bartha and that the two recombinants and stored at (cid:4)80°C. At the times of injection viral ali-
are capable of simultaneously co-infecting the same neu- quotswereremovedfromthefreezerandkeptoniceuntil
ronalpopulation(Standishetal.,1995;Billigetal.,1999, immediatelybeforeinjections.
2000, 2001; Smith et al., 2000; Kerman et al., 2003, Todeterminethelocationofneuronscoordinatingmus-
2006a). clecontractionandautonomicactivity,ratsreceivedinjec-
We previously reported a robust negative correlation tions of PRV-152 into their gastrocnemius muscles and
betweentheanimal’sweightandefficiencyofinfectionof injectionsofPRV-BaBluintotheiradrenalglands.Ineach
gastrocnemius motoneurons, with the largest rate of mo- animalthegastrocnemiusmusclewasinjectedwith30(cid:3)l
toneuroninfectioninratsofintermediatesizethatweigh ofPRV-152,whichwasdividedintomultipleinjectionsof
approximately200g(Kermanetal.,2003).Thus,animals 1.0 (cid:3)l. The adrenal gland was dissected via a left flank
in the current study were selected to weigh between 163 incision and gently separated from surrounding viscera
and 254 g for an average of 197 (cid:7) 8 g (mean (cid:7) SEM). andfat;theadjacentconnectivetissuewasthenretracted
Animalswereanesthetizedwitheitheraninhalableoran tofacilitateaccesstothegland.Twoorthreeinjectionsof
injectableanestheticprotocol.Intheformercaseanesthe- PRV-BaBlu were then made into the gland through a
sia was induced with 5% isoflurane vaporized in 1–1.5 glass pipette attached to a 10-(cid:3)l Hamilton syringe; the
L/min of O and maintained with a 1.5–2.5% concentra- totalvolumeofvirusinjectedwas2–4(cid:3)l.Followingeach
2
TheJournalofComparativeNeurology.DOI10.1002/cne
PSPMNSANDHYPOTHALAMICPEPTIDES 589
injection the gland was swabbed with a cotton-tip appli- jugated to CY3 (1:200; Jackson ImmunoResearch, West
cator to decrease nonspecific viral spread. We previously Grove, PA), goat anti-chicken IgG conjugated to Alexa
demonstrated that this injection protocol produces an ef- Fluor 488 (1:200; Molecular Probes Invitrogen, Carlsbad,
ficient infection of target neurons and does not result in CA), and goat anti-rabbit IgG conjugated to Alexa Fluor
spreadofvirustonon-targettissues(Kermanetal.,2003). 647(1:200;MolecularProbesInvitrogen)dissolvedinthe
Injected animals were assigned to one of three groups: blocking solution (1% NGS, 1% BSA, and 0.3% TX-100).
short,intermediate,andlongsurvivaltimes.Intheshort Following processing, tissue sections were mounted on
survival group (n (cid:2) 3) rats survived for 121–122 hours glassslidesandcoverslippedwithAquaPoly/Mount(Poly-
following injection with PRV-152 and for 96–97 hours sciences,Warrington,PA).
following PRV-BaBlu injection. We chose this survival
Tissue analysis
timepointbecausethisistheearliesttimepointatwhich
appreciable numbers of double-infected neurons are de- Images of antibody-labeled MCH and orexin-
tected in the rat hypothalamus (Kerman et al., 2006a). synthesizing neurons were taken at 10(cid:6) magnification
Because a single replication cycle of the virus is 10–12 with a flat-field correction by using a Zeiss Axiophot up-
hours (Demmin et al., 2001), we therefore decided to ex- right light microscope (Carl Zeiss MicroImaging, Thorn-
tend the survival times by 10–12 hours to determine the wood, NY), operated by a Ludl motorized stage that was
maximalnumberofdouble-infectedneuronsthatexpress connected to a Ludl MAC2000 XYZ controller module
MCH and orexins. Accordingly, in the intermediate sur- (Ludl Electronic Products, Hawthorne, NY). Images of
vival group (n (cid:2) 4) the animals survived for 132 or 136 individual fields of view were stitched together by using
and 108 or 112 hours following injections with PRV-152 the virtual slice tool in Stereo Investigator software (Mi-
and PRV-BaBlu, respectively. In the late survival group croBrightField,Williston,VT)onaDellPC.
(n (cid:2) 4) the animals survived for 143–144 and 111–120 Immunofluorescently labeled tissue was examined by
hoursfollowinginjectionswithPRV-152andPRV-BaBlu, using a Leica DMR photomicroscope (Wetzlar, Germany)
respectively. and with an Olympus FluoView 1000 laser scanning
confocal microscope (http://www.olympusfluoview.com/).
Tissue processing
Swanson’s (2004) rat atlas was used as a reference for
FollowingtranscardialperfusionwithPLP(seeabove), anatomical classification. When using Leica DMR micro-
theentirebrainwasextractedandpostfixedinPLPover- scope, the presence of each fluorophore was detected by
night. Brains were then immersed in 20% sucrose until usingspecificfiltersets(ChromaTechnology,Brattleboro,
they sank to the bottom of the container; they were sec- VT)withthefollowingrespectiveexcitationandemission
tionedcoronallyonafreezingmicrotomeatathicknessof ranges: Alexa Fluor 488, 440–520 nm and 500–555 nm
35or40(cid:3)mandcollectedintosixbins.Tissuewasstored (green fluorescence); CY3, 535–560 nm and 545–625 nm
at (cid:4)20°C in cryoprotectant until immunohistochemical (redfluorescence);andAlexaFluor647,560–680nmand
processingwasconducted. 625–775nm(farredfluorescence).
For immunofluorescent detection in the brain, tissue Adjacent black-and-white images of the LH were digi-
was initially rinsed with 0.1 M PB several times at room tized under a 20(cid:6) objective; images were then stitched
temperature. It was then incubated for 1 hour in the togetherbyusingthePhotomergefunctioninAdobePho-
blockingsolution(1%NGS,1%BSA,and0.3%TX-100in toshop CS (Adobe Systems, San Jose, CA). Each of these
0.1 M PB). Sections were then reacted with a cocktail of large stitched images corresponded to a different fluores-
primaryantibodies—chickenanti-GFPIgY(Abcam,prod- cent filter set and was pseudocolored as follows: GFP-
uct#13970)at1:2,000,mouseanti-(cid:1)-galIgG(Sigma,St. positive neurons, green; (cid:1)-gal-containing cells, red; and
Louis,MO,product#G4644)at1:1,000,andeitherrabbit MCH- or orexin-positive neurons, blue or cyan. These
anti-MCH at 1:10,000 or rabbit anti-orexin at 1:500— pseudocolored images were then overlaid, and co-
diluted in the above blocking solution. In each animal localization of two or three fluorophores was determined
adjacenttissuebinswereprocessedfororexin,GFP,(cid:1)-gal byturningeachlayeronandofftodeterminelocationand
triple-labeling and for MCH, GFP, (cid:1)-gal triple-labeling. colorofeachcell.Brightnessandcontrastofimageswere
Thechickenanti-GFPantibodywasraisedagainstrecom- optimizedforpresentationpurposes,andfigureswerepre-
binant full-length protein. This antibody yields a single paredinPhotoshop.
bandonWesternblotanddetectsGFPintransgenicmice
Statistical analyses
expressingGFPinlaminaIIofthespinalcord(manufac-
turer’s technical information). We have previously deter- Potential differences in distribution of labeled neurons
minedabsenceofstainingwiththisantibodyinuninfected between perifornical and peripeduncular regions were
rat tissue sections (Kerman et al., 2006b). Mouse anti-(cid:1)- evaluated with repeated measures ANOVA with region
gal antibody was developed in mouse peritoneal cavities (perifornical vs. peripeduncular) and anatomical level as
by using (cid:1)-gal purified from E. coli as the immunogen. independent variables and percentage of cells that were
UsingWesternblot,thisantibodywasshowntobespecific eitherMCH-positivePSPMNsororexin-positivePSPMNs
for (cid:1)-gal in its native form (116 kDa), and it reacts only asadependentvariable.Statisticalanalyseswerecarried
with(cid:1)-galfromE.coli(manufacturer’stechnicalinforma- outinStatView5.0(SASInstitute,Cary,NC),andsignif-
tion). The specificity of this antibody in immunofluores- icancewassetasP(cid:8)0.05.
cent experiments has been previously documented (Ker-
manetal.,2003). RESULTS
Following 48-hour incubation at 4°C in the primary
Specificity of the MCH antibody
antibody solution, the tissue was rinsed several times
with0.1MPBandthenreactedwithasecondaryantibody To confirm the specificity of the MCH antibody, we
solution, which consisted of donkey anti-mouse IgG con- performedablockingstudywiththespecificMCHpeptide.
TheJournalofComparativeNeurology.DOI10.1002/cne
590 I.A.KERMANETAL.
neurons in LH. Note the clustering of neurons in the
perifornical and peripeduncular regions, which is espe-
cially prominent at the caudal level that we examined
(Fig.2A).
Although orexin-positive neurons occupied a similar
area within LH, it was apparent that the bulk of these
neurons was located more medially than the MCH-
positive cells. Figure 2D–F illustrates the distribution of
orexin-positive cells; note that these neurons are prefer-
entially distributed within the perifornical region com-
paredwiththeperipeduncularregion.Withintheperifor-
nicalregion,orexin-positivecellsalsoappearedtobemore
prevalentthantheMCH-positivecells(comparethelabel-
inginFig.2FwiththatinFig.2C).
Expression of MCH within PSPMNs
In line with our previous observations (Kerman et al.,
2006a), we detected considerable numbers of double-
infected neurons within LH and DMH across all three
survival groups. Within LH these neurons were equally
distributed throughout the nucleus and were as likely to
be located in the perifornical region as in the peripedun-
cularregion.
We conducted triple-labeling experiments aimed at de-
termining whether hypothalamic PSPMNs expressed
MCH. In these experiments MCH-positive neurons were
taggedwithafarredfluorophoreandwerepseudocolored
cyan.Cellsthatsendpolysynapticprojectionstohindlimb
muscle were tagged with a green fluorophore, and those
that send polysynaptic projections to the adrenal gland
weretaggedwitharedfluorophore.Thus,double-infected
neuronsappearedyellow,whereastriple-labeledneurons
appearedasamixtureofallthreecolors.
Figure 3 illustrates examples of neurons infected with
PRV-152(green),orPRV-BaBlu(red),orboth(yellow)and
their relationship to the MCH-synthesizing neurons
(cyan).WedetectedMCHwithinPSPMNsthroughoutLH;
Fig. 1. Characterization of the MCH antiserum. Specificity was
ascertainedbyperformingablockingstudy.Adjacentsectionswere however, these triple-labeled cells appeared to be more
incubatedwiththeMCHantibodyalone(A)orwithMCHantibody numerous within the peripeduncular region (Fig. 3D2)
preincubatedwithMCHpeptide(B).NoterobustlabelinginAand thanwithintheperifornicalregion(Fig.3D1).
absenceoflabelinginB,indicatingspecificityoftheantibodybinding. We then mapped the locations of MCH-positive
Scalebar(cid:2)50(cid:3)minB(appliestoA,B).
PSPMNs, as well as the locations of neurons that were
infected with only one PRV strain but also expressed
MCH.Thesemapswerecreatedatthreedifferentrostro-
Inthisexperiment,sectionsthroughtheLHwerereacted caudallevelsoftherathypothalamusandacrossthethree
withtheanti-MCHantibodywithandwithoutpreincuba- different survival periods used in the current study. Fig-
tionofMCHpeptide.Undercontrolconditionswedetected ure4illustratesthedistributionsofthesecellsatdifferent
robust labeling in LH around the cerebral peduncle (Fig. survival times. Note that at the earliest survival time
1A).TheobservedMCHantibodylabelingwascompletely there are considerable numbers of PSPMNs in LH and
abolished by preincubation with the MCH peptide (Fig. DMH (Fig. 4A). Some of these neurons express MCH
1B), thus confirming the specificity of this antibody for (black circles) and are intermingled with MCH-negative
MCH. PSPMNs (yellow squares). Furthermore, these neurons
are present around the fornix (in the perifornical region)
Distribution of MCH- and orexin-
and around the cerebral peduncle (in the peripeduncular
synthesizing neurons
region).
Distribution of MCH- and orexin-synthesizing neurons With increased survival time, the numbers of detected
was studied in sections from adjacent (1 in 6 series) bins double-infected neurons increased and these cells were
that were visualized by using DAB as the chromogen. present throughout LH and DMH. However, with these
These studies demonstrated that although MCH-positive longer survival times MCH-positive PSPMNs (black cir-
neuronsweredistributedthroughoutLH,theyformedtwo cles)segregatedpreferentiallytowardtheperipeduncular
clear groupings. The first cluster of MCH neurons was region(Fig.4B,C).
located around the fornix (perifornical region), whereas
Expression of orexins within PSPMNs
the second cluster of these cells was found around the
cerebral peduncle (peripeduncular region). Figure 2A–C We conducted triple-labeling experiments to determine
demonstrates this distribution pattern of MCH-positive whether hypothalamic PSPMNs are orexinergic. In these
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PSPMNSANDHYPOTHALAMICPEPTIDES 591
Fig.2. DistributionofMCH-andorexin-positiveneurons.Distri- especiallypronouncedatthecaudalmostlevel(A).Althoughorexin-
bution of these neurons was examined at three rostrocaudal levels: ergicneuronsaredistributedthroughoutthelateralhypothalamus,
approximately(cid:4)3.80mm(A,D;level1),(cid:4)3.25mm(B,E;level2),and they tend to cluster in the perifornical region. This is especially
(cid:4)2.50 mm (C,F; level 3) from bregma. MCH-positive neurons are prominentattherostralmostlevel(F).Abbreviations:3v,thirdven-
shownontheleft(A–C),andorexin-positiveneuronsareshownonthe tricle;cpd,cerebralpeduncle;fx,fornix;mtt,mammillothalamictract;
right(D–F).NotetheclusteringofMCH-positiveneuronsaroundthe ot,optictract.Scalebar(cid:2)300(cid:3)minD(appliestoA–F).
fornix and at the medial edge of the cerebral peduncle, which is
experimentsorexin-positiveneuronsweretaggedwithafar As with the MCH-positive neurons, we mapped the lo-
redfluorophoreandpseudocoloredcyan.Neuronsthatsend cations of orexin-positive PSPMNs in relation to the
polysynaptic projections to hindlimb muscle were tagged orexin-negative double-infected neurons as well as to
withagreenfluorophore,whereasthosesendingpolysynap- orexin-positivesingle-infectedneurons(Fig.6).Thismap-
ticprojectionstotheadrenalglandweretaggedwithared pingrevealedthatorexin-positivePSPMNsweredetected
fluorophore. Figure 5 illustrates data from these experi- evenattheearliestsurvivaltimepointandthatthenum-
ments;notethatintheseexamplesthenumbersoforexin- bersofdouble-infectedcellsincreasedwithlongersurvival
positive PSPMNs are greater within the perifornical area times.Furthermore,asthenumbersofPRV-infectedneu-
(Fig.5D1)comparedwiththeperipedunculararea(Fig.5D2). rons increased with increasing survival, orexin-positive
TheJournalofComparativeNeurology.DOI10.1002/cne
592 I.A.KERMANETAL.
Fig. 3. Co-localization of viral reporter genes with melanin- colored with red (C1,C2). D1,D2: Merged images of A1–C2.
concentrating hormone (MCH) in neurons of the lateral hypothala- A1–D1: Images from the perifornical region (note the edge of the
mus.Triple-labeledsectionswereimagedwithalaser-scanningcon- fornixoutlinedwithadashedline).A2–D2:Imagesfromtheperipe-
focalmicroscopetaking40–60opticalsectioinsperfieldofview.For duncularregion.Triple-labeledneurons(MCH-positivePSPMNs)are
eachimagethez-stackwascompressed,andimageswererenderedfor shownwitharrows.AnMCH-positiveneuronthatwasinfectedonly
three-dimensional representation. A1,A2: MCH-positive neurons withPRV-BaBluisshownwiththickarrowsinA2–D2,andanMCH-
were tagged with Alexa Fluor 647 and pseudocolored with cyan. positiveneuronthatwasinfectedonlywithPRV-152isshownwith
B1,B2,C1,C2:Hypothalamicneuronsthatsendpolysynapticprojec- long narrow arrows in A2–D2. Double-infected neurons that are
tions to hindlimb muscle were infected with pseudorabies virus MCH-negativeareshownwitharrowheads.Notegreaternumbersof
(PRV)-152, tagged with Alexa Fluor 488, and pseudocolored with triple-labeledneuronsintheperipeduncularregion(2)comparedwith
green(B1,B2);neuronswithpolysynapticprojectionstotheadrenal theperifornicalregion(1).AbbreviationasinFigure2.Eachboxofthe
glandwereinfectedwithPRV-BaBlu,taggedwithCY3,andpseudo- gridis32(cid:3)m.
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PSPMNSANDHYPOTHALAMICPEPTIDES 593
Fig.4. DistributionofMCH-andPRV-containingneuronsinthe ing was examined at three rostrocaudal levels (similar to the ones
hypothalamus.Anatomicallandmarksweretracedfromoriginalsec- representedinFigure2:1,caudal;2,intermediate;3,rostral.Panels
tions, and locations of virus-infected cells were plotted onto these ontheleft(A1–A3)arefromananimalintheshortsurvivalgroup,
maps. Only one hemisection is presented from each level, but the thoseinthemiddle(B1–B3)arefromananimalintheintermediate
pattern of labeling was symmetrical. Each symbol represents one survivalgroup,andthoseontheright(C1–C3)arefromananimalin
neuronontheactualsection.Yellowsquaresrepresentneuronsco- thelongsurvivalgroup.Notethatalthoughdouble-infectedneurons
infectedwithPRV-152(polysynapticprojectionstohindlimbmuscle) are distributed throughout the lateral and dorsomedial hypothala-
and PRV-BaBlu (polysynaptic projections to adrenal gland). Black mus,PSPMNsthatexpressMCHarepreferentiallydistributedinthe
circlesrepresentneuronsco-infectedwithPRV-152andPRV-BaBlu peripeduncularregion(alongthemedialedgeofcerebralpeduncle).
that also express MCH. Red triangles represent MCH-containing Thisisespeciallyprominentatlongersurvivaltimepoints(forexam-
neuronsthatareinfectedonlywithPRV-BaBlu,andaquadiamonds ple,C2).AbbreviationsasinFigure2.
indicateMCH-containingneuronsinfectedonlywithPRV-152.Label-
PSPMNs preferentially clustered within the perifornical tively.Thisclusteringisapparentatintermediate(Fig.7B)
regionofLH(Fig.6B,C). andlong(Fig.7C)survivaltimepoints.
Todeterminewhetherthissegregationintomedialand
Comparison of the distribution of MCH- and
lateral groups of orexin- and MCH-positive PSPMNs is
orexin-positive PSPMNs
true throughout the rostrocaudal extent of LH, we per-
To compare the distribution of MCH- and orexin- formed the same mapping procedure at three different
positive PSPMNs directly, double-infected neurons that rostrocaudallevelsfordatacollectedfromtwoofthelong-
werepositiveforeachofthesepeptidesweremappedonto surviving animals. Figure 8 illustrates results from this
correspondingatlasplatesfromSwanson’s(2004)ratbrain study; these data also indicate segregation of MCH- and
atlas.Figure7illustratesdatafromthismappingstudy;at orexin-positive PSPMNs into lateral and medial groups
eachsurvivaltimepointMCH-andorexin-positivePSPMNs withinLH.
wereprojectedontothesameatlasplate(plate#30inSwan-
Quantification
son’s(2004)ratbrainatlas.Thesedatademonstrateaclear
segregationofMCH-(reddots)andorexin-(bluedots)syn- Todetermineregionaldifferencesinthedistributionof
thesizingPSPMNsintolateralandmedialclusters,respec- PRV-infected neurons, we quantified the numbers of: 1)
TheJournalofComparativeNeurology.DOI10.1002/cne
594 I.A.KERMANETAL.
Fig.5. Co-localizationofviralreportergeneswithorexininneu- CY3,andpseudocoloredwithred(C1,C2).D1,D2:Mergedimagesof
ronsofthelateralhypothalamus.Triple-labeledsectionswereimaged A1–C2.A1–D1:Imagesfromtheperifornicalregion(notetheedgeof
withalaser-scanningconfocalmicroscopetaking40–60opticalsec- the fornix outlined with a dashed line). A2–D2: Images from the
tionsperfieldofview.Foreachimagethez-stackwascompressed, peripeduncular region. Triple-labeled neurons (orexin-positive
and images were rendered for three-dimensional representation. PSPMNs)areshownwitharrows.Double-infectedneuronsthatare
A1,A2: Orexin-positive neurons were tagged with Alexa Fluor 647 orexin-negativeareshownwitharrowheads.ThickarrowsinA1–D1
and pseudocolored with cyan. B1,B2,C1,C2: Hypothalamic neurons andA2–D2showorexin-positiveneuronsthatwereinfectedonlywith
thatsendpolysynapticprojectionstohindlimbmusclewereinfected PRV-BaBlu. Note greater numbers of triple-labeled neurons in the
withpseudorabiesvirus(PRV)-152,taggedwithAlexaFluor488,and perifornicalregion(1)comparedwiththeperipeduncularregion(2).
pseudocoloredwithgreen(B1,B2);neuronswithpolysynapticprojec- Images were taken from the same animal as shown in Figure 4.
tionstotheadrenalglandwereinfectedwithPRV-BaBlu,taggedwith AbbreviationasinFigure2.Eachboxofthegridis32(cid:3)m.
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PSPMNSANDHYPOTHALAMICPEPTIDES 595
Fig.6. Distributionoforexin-andPRV-containingneuronsinthe indicateorexin-containingneuronsinfectedonlywithPRV-152.La-
hypothalamus.Anatomicallandmarksweretracedfromoriginalsec- belingwasexaminedatthreerostrocaudallevelssimilartotheones
tions, and locations of virus-infected cells were plotted onto these representedinFigure2:1,caudal;2,intermediate;3,rostral.Panels
maps. Only one hemisection is presented from each level, but the ontheleft(A1–A3)arefromananimalintheshortsurvivalgroup,
pattern of labeling was symmetrical. Each symbol represents one thoseinthemiddle(B1–B3)arefromananimalintheintermediate
neuronontheactualsection.Yellowsquaresrepresentneuronsco- survivalgroup,andthoseontheright(C1–C3)arefromananimalin
infectedwithPRV-152(polysynapticprojectionstohindlimbmuscle) thelongsurvivalgroup.Notethatalthoughdouble-infectedneurons
and PRV-BaBlu (polysynaptic projections to adrenal gland). Black are distributed throughout the lateral and dorsomedial hypothala-
circlesrepresentneuronsco-infectedwithPRV-152andPRV-BaBlu mus, orexin-positive PSPMNs are preferentially distributed in the
that also express orexin. Red triangles indicate orexin-containing perifornicalregion(e.g.,inC2).AbbreviationsasinFigure2.
neuronsthatareinfectedonlywithPRV-BaBlu,andaquadiamonds
double-infected neurons that were MCH- or orexin-nega- atlas, and symbols were counted within the different hy-
tive;2)double-infectedneuronsthatwereMCH-ororexin- pothalamicnuclei(Tables1,2).BasedonSwanson’satlas,
positive;and3)neuronsthatwereinfectedwithonlyone thefollowingLHsubnucleiweregroupedtogethertorep-
of the PRV strains and were either MCH- or orexin- resent the peripeduncular region: dorsal region; ventral
positive.Thisprocedurewascarriedoutintissueobtained region, lateral zone; ventral region, medial zone; magno-
fromthreeratsfromtheintermediatesurvivaltimepoint. cellularnucleus;andparvicellularregion.Inaddition,the
Thissurvivaltimewaschosenbecauseitisthefirsttime preparasubthalamic nucleus was also included in the
point at which appreciable numbers of double-infected peripeduncular region. For the perifornical region, the
neuronsaredetectedthatprobablysenddirectprojections posterior hypothalamus was included as well as the fol-
to the spinal cord. Quantification was performed on sec- lowing LH subnuclei: juxtadorsomedial; suprafornical;
tionsfromthreedifferentanatomicallevels,asillustrated juxtaventromedial region, dorsal zone; and juxtaventro-
inFigure2. medialregion,ventralzone.
Digitizedtissuesectionsweremarkedupwithdifferent To evaluate potential differences in the distribution of
symbolsrepresentingthesecombinationsofmarkers(see MCH-positive PSPMNs between perifornical and peripe-
Figs. 4 and 6 for examples). These maps were then pro- duncularregions,cellcountsforeachanatomicallevelin
jected onto digital plates from the Swanson’s (2004) rat each animal were expressed as percentage of total neu-