Arousal Feedforward Circuit That May Enhance Cognitive Paraventricular Nucleus-Excited by ...

14 Pages·2006·0.67 MB·English

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Prefrontal Cortex Projecting Glutamatergic Thalamic
Paraventricular Nucleus-Excited by Hypocretin: A
Feedforward Circuit That May Enhance Cognitive
Arousal
Hao Huang, Prabhat Ghosh and Anthony N. van den Pol
J Neurophysiol 95:1656-1668, 2006. ;
doi: 10.1152/jn.00927.2005
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Journal of Neurophysiology publishes original articles on the function of the nervous system. It is published 12
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Copyright © 2006 by the American Physiological Society. ISSN: 0022-3077, ESSN: 1522-1598. Visit our website
at http://www.the-aps.org/.
JNeurophysiol95:1656–1668,2006;
doi:10.1152/jn.00927.2005.
Prefrontal Cortex–Projecting Glutamatergic Thalamic Paraventricular
Nucleus-Excited by Hypocretin: A Feedforward Circuit That May
Enhance Cognitive Arousal
Hao Huang, Prabhat Ghosh, and Anthony N. van den Pol
DepartmentofNeurosurgery,YaleUniversitySchoolofMedicine,NewHaven,Connecticut
Submitted7September2005;acceptedinfinalform4November2005
Huang, Hao, Prabhat Ghosh, and Anthony N. van den Pol. (PengandBentivoglio2004)andfiberscontainingNO(Otake
Prefrontal cortex–projecting glutamatergic thalamic paraventricular and Ruggiero 1995), which may modulate sleep and arousal D
nucleus-excited by hypocretin: a feedforward circuit that may en- o
(Aston-Jonesetal.2001;Cespuglioetal.1998;Williamsetal. w
hance cognitive arousal. J Neurophysiol 95: 1656–1668, 2006; n
doi:10.1152/jn.00927.2005. The paraventricular thalamic nucleus 1997). In addition, PVT receives inputs from homeostatic lo
(PVT) receives one of the most dense innervations by hypothalamic controlregionsofthebrainincludingthenucleusofthesolitary ad
e
hypocretin/orexin(Hcrt)neurons,whichplayimportantrolesinsleep- tract (Otake and Ruggiero 1995), parabrachial nucleus (Krout d
wakefulness,attention,andautonomicfunction.ThePVTprojectsto and Loewy 2000), and from many regions of hypothalamus fro
m
severalloci,includingthemedialprefrontalcortex(mPFC),acortical (ChenandSu1990;CornwallandPhillipson1988;Otakeand
h
region involved in associative function and attention. To study the Ruggiero 1995). ttp
eufsfeedctaofnHewcrtloinneexocfitattroarnysgPeVniTcnmeuircoensexthpartespsrionjgectgtroeetnheflmuPoFreCsc,ewnet PVTefferentsareuniqueamongthemidlinethalamicnuclei ://jn
protein (GFP) under the control of the vesicular glutamate-trans- and project to the ventral aspects of medial prefrontal cortex .p
porter-2 promoter. These neurons were retrogradely labeled with (mPFC), particularly the infralimbic and prelimbic cortices, hy
s
cholera toxin subunit B that had been microinjected into the mPFC. nucleus accumbens, and amygdala (Berendse and Groenewe- io
Membrane characteristics and responses to hypocretin-1 and -2 gen 1991; Bubser and Deutch 1998; Conde et al. 1990; Moga log
(Hcrt-1 and -2) were studied using whole cell recording (n (cid:1) 300). et al. 1995; Su and Bentivoglio 1990), all of which are y.o
PVTneuronsshoweddistinctmembranepropertiesincludinginward associated with limbic function including motivation and at- rg
rectification, H-type potassium currents, low threshold spikes, and tention(Cardinaletal.2002;Christakouetal.2004).Manyof a/
spikefrequencyadaptation.Corticallyprojectingneuronsweredepo- these projections are excitatory (Christie et al. 1987; Frassoni t P
larizedandexcitedbyHcrt-2.Hcrt-2actionswerestrongerthanthose et al. 1997). Because the ventral parts of the mPFC play key en
of Hcrt-1, and the action persisted in TTX and in low calcium/high rolesinexecutiveaspectsofattentionandabroadspectrumof n S
magnesiumartificialcerebrospinalfluid,consistentwithdirectactions limbicandassociativefunctions,thatPVTneuronsthatproject ta
mediated by Hcrt receptor-2. Two mechanisms of Hcrt excitation te
were found: an increase in input resistance caused by closure of here are of particular interest. Thus the axonal projections are U
one characteristic that set PVT apart from other midline tha- n
potassium channels and activation of nonselective cation channels. iv
The robust excitation evoked by Hcrt-2 on cortically projecting lamic nuclei. o
n
glutamatePVTneuronscouldgeneratesubstantialexcitationinmul- Hypocretin/orexin (Hcrt) neurons, found exclusively in the F
tiplelayersofthemPFC,addingtothemoreselectivedirectexcitatory lateral hypothalamus and perifornical area (de Lecea et al. eb
actionsofHcrtinthemPFCandpotentiallyincreasingcorticalarousal 1998; Sakurai et al. 1998), have been implicated in the regu- ru
a
andattentiontolimbicorvisceralstates. lation of sleep-wakefulness, feeding, neuroendocrine, and au- ry
tonomicfunctions(FergusonandSamson2003;Sakurai2002). 23
Hcrtincreasesarousal(Haganetal.1999),whereasdisruption , 2
INTRODUCTION 0
of the Hcrt system leads to narcolepsy (Chemelli et al. 1999; 1
3
The paraventricular nucleus of the thalamus (PVT) is a Linetal.1999;Nishinoetal.2000).TwoHcrtpeptides(Hcrt-1
uniquemidlineintralaminarnucleusthathasbeenimplicatedin and Hcrt-2) are synthesized by the same neurons and act on
the regulation of autonomic and visceral functions (Bhatnagar two receptors (Hcrt-R1 and Hcrt-R2) (Sakurai et al. 1998).
and Dallman 1999; Van der Werf et al. 2002). PVT neurons Hcrt fibers and receptors show a widespread distribution
alsorespondtostressandtoperipherallyadministeredpsycho- throughoutthebrainandspinalcord,withhighlevelsinsome
stimulants cocaine and amphetamine (Bubser and Deutch selected brain regions, particularly the PVT (Marcus et al.
1999; Deutch et al. 1998). PVT receives heavy monoamine 2001; Peyron et al. 1998; van den Pol 1999).
inputs that include histamine, dopamine, noradrenaline, and High levels of Hcrt immunoreactive axons and receptors
serotonin fibers (Cornwall and Phillipson 1988; Otake and suggest the PVT is an important CNS site where Hcrt acts to
Ruggiero1995;Panulaetal.1989;RicoandCavada1998),all influence arousal, autonomic functions, and limbic activities.
of which have been implicated in the promotion and mainte- Thisstudyuseswholecellrecordingandanatomicaltracingin
nance of wakefulness (Jones 2003; Siegel 2004). PVT also transgenic mice that express green fluorescent protein (GFP)
receives inhibitory fibers from the suprachiasmatic nucleus under control of a glutamate vesicular transporter-2 (vGluT2)
Addressforreprintrequestsandothercorrespondence:A.N.vandenPol, Thecostsofpublicationofthisarticleweredefrayedinpartbythepayment
Dept.ofNeurosurgery,YaleUniv.,SchoolofMedicine,333CedarSt.,New ofpagecharges.Thearticlemustthereforebeherebymarked“advertisement”
Haven,CT06520(E-mail:[email protected]). inaccordancewith18U.S.C.Section1734solelytoindicatethisfact.
1656 0022-3077/06$8.00Copyright©2006TheAmericanPhysiologicalSociety www.jn.org
PREFRONTAL CORTEX-PVT ACTIVATION BY HYPOCRETIN 1657
promoter to examine Hcrt modulation of the glutamatergic constructed from a sigmoid function with Igor Pro software. The
PVT cells that project to the mPFC. restingmembranepotential(RMP)wasdeterminedinthefirst3min
ofwholecellrecording.TomeasuretheHcrt-inducedchangesinthe
input membrane resistance, short-lasting hyperpolarizing current
METHODS pulses were applied repetitively. In some experiments, at the maxi-
Preparation of thalamic slices mumamplitudeofthedepolarizingeffectcausedbyHcrt,themem-
branepotentialwasclampedmanuallybacktoitsrestingvalue,thus
Thalamic slices were prepared from nontransgenic control or allowingustoevaluatethechangeinmembraneinputresistance.
vGluT2-GFPtransgenicmicethatexpressenhancedGFPselectively
in presumptive glutamatergic neurons, as described previously for Vesicular glutamate transporter 2 drives GFP expression
hypothalamusslices(Lietal.2002).
in transgenic mouse
Briefly, 14- to 21-d-old mice maintained in a 12/12-h light/dark
cycle were given an overdose of pentobarbital sodium (100 mg/kg) Transgenic mice in which the vGluT2 promoter 1.8 kb upstream
duringthelightpartofthecycle(11:00AMto4:00PM),andthebrains fromthevGluT2sequencewereusedtodriveGFPexpression.Two
wereremovedrapidlyandplacedinice-coldoxygenated(95%O -5% short oligomers, one forward primer with sequences 5(cid:8)-ATCTC-
2
CO ) high-sucrose solution that contained (in mM) 220 sucrose, 2.5 GAGACGCACTCCCCCTGGTTGATTTAG-3(cid:8) and one reverse
KC2l,6MgCl ,1CaCl ,1.23NaH PO4,26NaHCO ,and10glucose, primer containing 5(cid:8)-CCGCGGTACCTCTTGTAAAGACTGGTGT- D
2 2 2 3 o
pH 7.4 (when equilibrated with a mixture of 95% O -5% CO ). A CCAGCCTTACCAGATTTA-3(cid:8)correspondingtotheaminoterminal w
thalamicblockwasprepared,andcoronalslices(220–2300(cid:1)mt2hick) region immediately upstream of the mouse vGluT2 coding region nlo
werecutonavibratome.Aftera1-to2-hrecoveryperiod,sliceswere were used to synthesize a 1.8-kb PCR fragment from mouse whole ad
moved to a recording chamber mounted on a BX51WI upright braingenomicDNAtemplate.This1.8-kbPCRfragmentwascloned ed
microscope(Olympus,Tokyo,Japan)equippedwithvideo-enhanced in the TA-TOPO II cloning system, and the sequence was verified. fro
infrared-differential interference contrast (DIC) and fluorescence. The1.8-kbpromoterfragmentwascutoutfromtheTOPOIIplasmid m
Slices were perfused with a continuous flow of gassed artificial by digestion with restriction enzymes XhoI and KpnI, purified and h
cerebrospinalfluid(ACSF;95%O -5%CO )thatcontained(inmM) subsequentlyclonedatXhoIandKpnIsitesintoapEGFP-1plasmid ttp
124NaCl,2.5KCl,2MgCl2,2Ca2Cl2,1.232NaH2PO4,26NaHCO3, lacking a promoter. The constructed vGluT2-GFP plasmid was di- ://jn
and10glucose,pH7.4.Experimentsinwhichequilibriumpotentials gestedwithBspH1andAfe1toisolatethe3,369-bpfragment,which .p
forpotassium(EK)waschangedweredonewiththefollowingACSF contains the mouse vGluT2 promoter followed by GFP coding se- hys
(inmM):111.5NaCl,15KCl,2MgCl2,2CaCl2,1.23NaH2PO4,26 quences and SV40 poly(A) signal sequences. This 3.3-kb DNA io
NaHCO ,and10glucose.AccordingtotheNernstequation,E was fragment was purified and prepared for microinjection to generate lo
(cid:2)108.33mV in control ACSF ([K] (cid:3) 2.5 mM) and (cid:2)60.5 mKV in vGluT2transgenicmice,usingmethodspreviouslydescribed(vanden gy
modifiedACSF([K]o(cid:3)15mM).Tiossueswerecontinuouslyperfused Pol and Ghosh 1998; van den Pol et al. 2002, 2004). In these mice, .org
wmiatihntAaiCneSdFatso3l5ut(cid:4)ion1.°CThuesinAgCaSdFuatel-mchpaenranteulrheeaintctohnetrcohllaemr(bWerarwnaesr GbrFaiPn-etxhpatrepsrseinvgiounseluyrohnasvewbeereenfosuhnodwnontloyeixnprseeslescvteGdlurTeg2i,oinnscloufditnhge at P/
Instruments,Hamden,CT).NeuronswerevisualizedwithanOlympus thePVT(Fremeauetal.2001). e
Optical (cid:5)40 water-immersion lens. The Yale University Committee nn
onAnimalCareandUseapprovedallproceduresusedinthisstudy. S
Identification of PVT neurons that project to the ta
prefrontal cortex te
Patch-clamp recording U
Mice were anesthetized, and the retrograde tracer cholera toxin niv
Wholecellcurrent-andvoltage-clamprecordingswereperformed subunitB(CTB,AlexaFluor594conjugate,0.5%,400nl,dissolved o
using pipettes with 4- to 6-M(cid:6) resistance after being filled with insterilePBS)wasinjectedintothemPFCareathroughaglasspipette n F
pipettesolution.Thepipettesweremadeofborosilicateglass(World (tipdiameter,20–40(cid:1)m)attachedtoaHamiltonsyringe.Aftera5- e
b
Precision Instruments, Sarasota, FL) using a PP-83 vertical puller to7-daysurvivalperiod,theanimalswereanesthetizedandperfused ru
I(t2phPNoeSMtaeCprngiisstp-hi)Aea,itlg1TteecPM,os,roTgraleCouncktldti2yio,oo0n1n,.05swJHawaNpsEeaaarPn2es-E)G.fdSooFT,lnlo1Poer.,w1ompsEfHof(G-islntTi7nm.Are3e.Mc(wSoo)rlri:odt11hwi0n4gK5BasnOKA,dHtMPhfT.eaesALSctO,iocqamo4usmi(pidonoprdsejKiuintcnisCaocatnltteiifdoooo)nnfr, wcorbeunrisattcihganeet4nsl2%taw0tmie–pnir3aicezr0reaoud(cid:1)fssocemrsodmlpiodtaynoel.dsapA,erfhderTyeprdeieaexezrdeaii,nsnsgcrlPoiemcBvdeieSscfir.rslaoCtlseitpoordprmweoesendac,as,raliuanbsnsedeedcddsteieatocxobntaoisdmoveoneitfsneatecwhndtaedClruaewTsmimBenur.goesOuwfluntushteeeoerdde-r ary 23, 2013
ofthecapacitancewereautomaticallyperformedusingPulsesoftware forwholecellrecording.
(HEKA Elektronik, Lambrecht/Pfalz, Germany). Access resistance
wascontinuouslymonitoredduringtheexperiments.Onlythosecells Immunocytochemistry
inwhichaccessresistance(changes(cid:7)10%)wasstablewereincluded
intheanalysis.AnEPC9amplifierandPulsesoftwarewereusedfor Immunostaining was performed as described previously (van den
data acquisition (HEKA Elektronik). PulseFit (HEKA Elektronik), Pol 1999; van den Pol et al. 1998). Briefly, mice were anesthetized
Axograph(Axoninstruments,FosterCity,CA),andIgorPro(Wave- andperfusedtranscardiallywithafixativecontaining4%paraformal-
Metrics, Lake Oswego, OR) software were used for analysis. Both dehydein0.1Mphosphatebuffer.Sectionswerecutonacryostatat
excitatory and inhibitory spontaneous postsynaptic currents were 20–30 (cid:1)m (or 16 (cid:1)m for counting immunoreactive Hcrt axons and
detectedandmeasuredwithanalgorithminAxograph,andonlythose boutons).Afterwashinginnormalbuffercontaining0.1%lysine,1%
events with amplitude (cid:1)5 pA were used, as described in detail bovine serum albumin, 1% normal goat serum, and 0.3% Triton
previously (Gao and van den Pol 1999). The frequency of action X-100, sections were incubated overnight in primary guinea pig
potentials was measured using Axograph as well. The response to antiserum against vGluT2 (Chemicon International, Temecula, CA;
Hcrtwasconsideredtobepositiveifthemeanfiringratewasgreater diluted 1:2,500), rabbit antiserum against the neuroactive peptide
than(cid:4)20%ofcontrol,orachangeinmembranepotentialwasgreater Hcrt-2, or rabbit antiserum against GFP. Specificity of Hcrt-2 anti-
than (cid:4)3 mV. Data are expressed as means (cid:4) SE. Group statistical serum is described elsewhere (van den Pol 1999; van den Pol et al.
significance was assessed using Student’s t-test, paired Student’s 1998). A secondary antiserum of donkey anti-guinea pig or goat
t-test, and one-way ANOVA. Concentration–response curves were anti-rabbit immunoglobulin conjugated to Texas red (1:200; Molec-
JNeurophysiol(cid:127)VOL95(cid:127)MARCH2006(cid:127)www.jn.org
1658 H. HUANG, P. GHOSH, AND A. N. VAN DEN POL
ularProbes)wasused.Insomeexperiments,sectionswereincubated tive neurons in the thalamus and hypothalamus. Substantially
in biotinylated goat anti-rabbit Ig, washed, and treated with avidin- lessGFPexpressionwasfoundincorticalareaswherevGluT1
biotin-peroxidase complex (Vector Laboratories, Burlingame, CA).
is the primary vesicular glutamate transporter (Fremeau et al.
Sectionswerereactedwithdiaminobenzidineandhydrogenperoxide
2001; Herzog et al. 2001). Figure 1C shows GFP-immunore-
torevealthehorseradishperoxidasecomplex.
activeneuronsandterminalsinPVT.Figure1,D–F,showsthe
distribution of vGluT2-GFP–positive neurons in the anterior,
Quantification of immunoreactive Hcrt axons and boutons
central,andposteriorpartsofthePVT.TheexpressionofGFP
Immunoreactive Hcrt axons and boutons were quantified with a drivenbythevGluT2promoterwasconsistentwiththepattern
(cid:5)40 objective, combined with an additional (cid:5)1.5 magnification. of in situ hybridization showing vGluT2 mRNA in the PVT
Threedifferentsectionswereusedforeachbrain,andthenumberof
(Fremeau et al. 2001; Herzog et al. 2001; Hur and Zaborszky
axons and boutons were counted in three different areas for each
2005).SinglecellRT-PCRwasusedtoconfirmthatgreencells
section. The density of axons and boutons were studied in fields of
7,500 (cid:1)m2. Both single terminal boutons and boutons en passant expressedvGluT2.AllsixGFP-containingneuronsinthePVT
alongthefiberswerecounted.Therelativedensityofimmunoreactive aspirated into recording pipettes showed positive mRNA ex-
axons was determined by counting axon intersections with a super- pression.
D
imposedtestgridplacedoverthehistologicalimageoftheaxons.The AfterimmunocytochemicalstainingforthevGluT2antigen, o
testgridconsistsofthreeparallellinesandthreelinesperpendicular the distribution of vGluT2 immunoreactive axons in the PVT w
n
tothose.HcrtimmunoreactivePVTandlocuscoeruleussectionscut and also in the mPFC was examined. Figure 1G shows the lo
a
atthesamethicknessfromthesamemicewerecompared. vGluT2-immunostaining in the PVT. In the mPFC, vGluT2 d
e
Drugs and drug application ilmaymerusnIo,reVa,ctainvde VaxI,onaanld baopupteoanrsslaigrehtldyelnesseslydednissetriibnudteedepeinr d fro
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didDel-2(-BamICi)n,o-65--cpyhaonsop-h7o-nnoitproenqtuainnooixcaalicnied-2(A,3P-d5i)o,nbeicu(cCuNllQinXe)m, e(t(cid:4)hi)o–- lvaGyelurTV2I-imbmthuannoVstaIianin(dgatiannlaoytesrhVowonf).thFeigmuPreFC1H. shows the http
amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide ://jn
(AMPA),N-methyl-D-aspartate(NMDA),5-aminomethyl-3-hydroxy- .p
Membrane properties of vGluT2-GFP–positive neurons h
isoxazole (muscimol), and l-glutamate were purchased from Sigma y
s
(St.Louis,MO).Hcrt-1/orexinA(mouse)wasobtainedfromPhoenix io
Pharmaceutical. Hcrt-2 was synthesized by the Stanford University TheactiveandpassivemembranepropertiesofPVTneurons lo
PeptideFacility.TTXwasobtainedfromAlomoneLabs(Jerusalem, havenotbeensubstantivelycharacterized.Ourfirstgoalwasto gy
Israel).CTBwasobtainedfromMolecularProbes(Eugene,OR).All examine the membrane properties of vGluT2-GFP–positive .org
drugsweregivenbyflowpipetteapplication,directedattherecorded PVT neurons. In vGluT2-GFP–positive PVT neurons, cells a/
cell,unlessotherwisenoted.Drugsolutionswerepreparedbydiluting were either quiescent or fired regular spontaneous action po- t P
theappropriatestocksolutionwithACSF,unlessotherwisenoted. tentials(activecells)withameanfiringrateforactivecellsof e
n
1.52 (cid:4) 0.21 Hz (n (cid:3) 33; Table 1). The RMP was 57.9 (cid:4) 0.4 n
RESULTS mV (n (cid:3) 103), and the mean input resistance was 465 (cid:4) 18 Sta
m(cid:6) (n (cid:3) 25). As reported previously (Ishibashi et al. 2005), te
Strong Hcrt axon innervation of PVT U
the spontaneous action potentials were followed by an after- n
The PVT receives axonal input from hypothalamic Hcrt hyperpolarization (AHP; Fig. 2B) and were TTX-sensitive iv o
neurons (Peyron et al. 1998). To get a general perspective of (data not shown), suggesting that they are sodium-mediated n
F
the level of innervation, we compared the density of Hcrt action potentials. The mean input resistance was calculated e
b
immunoreactive boutons and axons in the PVT with the locus from the slope of the current-voltage relation. In all neurons ru
cleoveerlusleoufs,Hacbrtraiinnnreergviaotinocnoinnsitdheerebdratoinha(Hveorovnaethofetthael.hi1g9h9e9s;t s(cid:2)tu5d0iemd,Vthaendcusrhreonwt-evdoaltatgimeer-eilnadtieopnenwdaesntli(nFeiagr.b2e,twA1eeannd0 Aan2d) ary 2
PHecyrtrotneremtianl.al1s99in8).thFeigPuVreT1,aAndanLdCB.,Tshhoewmimeamnudneonresiatcytivoef iinndwiacradtinrgecttihfiecaetxioisntebnecteweoefnin(cid:2)w5a0rdlayndre(cid:2)ct8if0yinmgVK((cid:10)Figc.on2dAu3c)-, 3, 20
1
immunoreactive Hcrt axons was similar in the two regions, tance.AsshowninFig.2A2˙andTable1,mostoftheneurons 3
with a mean density of 67 (cid:4) 5 and 73 (cid:4) 4 grid intersections/ displayedamembranerectificationcharacterizedbyasagthat
fieldinPVTandLC,respectively(n(cid:3)4,P(cid:1)0.1).Themean has been identified as a time- and voltage-dependent rectifica-
density of immunoreactive Hcrt boutons in PVT and LC was tionresultingfromthepresenceofanH-typepotassiumcurrent
81 (cid:4) 9 and 93 (cid:4) 11/field, respectively (n (cid:3) 4, P (cid:1) 0.1). (H-current)inotherneurons.Whentheneuronsweredepolar-
ized from a hyperpolarized level, all the neurons recorded
showedalow-thresholdspike(LTS;Fig.2C1)(Ishibashietal.
vGluT2-GFP transgenic mouse
2005; Richter et al. 2005). The LTS was calcium-dependent,
Previous work has suggested that a subpopulation ((cid:9)60%) because it always persisted in the presence of TTX but was
ofPVTneuronsusesglutamateasaneurotransmitter,whereas eliminated by nickel at 200 (cid:1)M (n (cid:3) 6; Fig. 2, C2–C4). To
otherPVTneuronsuseunidentifiedtransmitters(Christieetal. determine the effect of current injection on action potentials,
1987; Frassoni et al. 1997). Three vesicular glutamate trans- weexaminedtheeffectofalong(3s)square-wavepulseof40
porters have been cloned (Fremeau et al. 2004), and in situ and120pA.AsshowninTable1andFig.2,D1andD2,most
hybridizationstudieshaveshownthatglutamatergiccellsinthe ofthePVTneuronsdisplayedclearspikefrequencyadaptation
PVTexpressvGluT2(Fremeauetal.2001;Herzogetal.2001; (SFA; the frequency of spikes is reduced over time) or spike
Hur and Zaborszky 2005). We used a transgenic mouse that failureathighercurrents.Asmallnumber(5of19)ofneurons
showedstrongGFPexpressioninsubpopulationsofpresump- showed only modest SFA (Fig. 2, E1 and E2; Table 1).
JNeurophysiol(cid:127)VOL95(cid:127)MARCH2006(cid:127)www.jn.org
PREFRONTAL CORTEX-PVT ACTIVATION BY HYPOCRETIN 1659
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FIG.1. Glutamate vesicular transporter-2 g
(vGluT2)-green fluorescent protein (GFP)–posi- y.o
tive neurons in the paraventricular thalamic nu- rg
cleus (PVT). A and B: hypocretin/orexin (Hcrt) a/
immunoreactive axons and boutons in PVT (A) t P
and locus coeruleus (LC) (B). C: peroxidase- e
labeledGFP-immunoreactiveneuronsandtermi- n
n
nalsinPVT.D–F:vGluT2-GFP–positiveneurons S
inanterior(D),central(E),andposterior(F)parts ta
ofPVT.3V,thirdventricle;IMD,intermediodor- te
sal nucleus. G and H: immunoreactive vGluT2 U
n
terminals in PVT (G) and layer V of medial iv
prefrontalcortex(mPFC)(H).Scalebar:A–C,15 o
(cid:1)m;D–F,50(cid:1)m;GandH,5(cid:1)m. n F
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JNeurophysiol(cid:127)VOL95(cid:127)MARCH2006(cid:127)www.jn.org
1660 H. HUANG, P. GHOSH, AND A. N. VAN DEN POL
TABLE 1. Properties of neurons in PVT
NontransgenicMice n vGluT2-GFP(cid:10)Neurons n vGluT2-GFP(cid:2)Neurons n
Restingpotential,mV (cid:2)58.1 (cid:4) 0.44 101 (cid:2)57.9 (cid:4) 0.41 103 (cid:2)59.4 (cid:4) 0.45 97
Inputresistance,M(cid:6) 430 (cid:4)14 12 465 (cid:4)18 25 437 (cid:4)17 31
Spikefrequency,Hz 1.41(cid:4) 0.24 48 1.52(cid:4) 0.21 33 1.46(cid:4) 0.38 30
Percentofcellsfiring 57%(48/84) 66%(33/50) 64%(30/47)
PercentofcellswithclearIh 71%(12/17) 69%(20/29) 54%(13/24)
PercentofcellswithclearSFA Nodatacollected 74%(14/19) 54%(13/24)
Activecells n Silentcells n
Restingpotential,mV (cid:2)58.4 (cid:4) 0.4 106 (cid:2)59.1 (cid:4) 0.6 68
Inputresistance,M(cid:6) 451 (cid:4)11 38 435 (cid:4)19 17
PercentofcellswithclearI 81%(26/32) 42%(5/12)
h
PercentofcellswithclearSFA 58%(14/24) 80%(8/10)
Valuesaremeans (cid:4) SD.Numbersinparenthesesrefertothenumberofcellswiththatproperty—thetotalnumberofcellstested.Ih,Hcurrent;SFA,spike D
frequencyadaptation. o
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To determine if the membrane properties of vGluT2-GFP– application of 50 nM Hcrt-2 and flow pipette application of 1 lo
a
positive cells differ from vGluT2-GFP–negative cells, we re- (cid:1)MHcrt-2bothdepolarizedandexcitedfiveinsevenneurons de
d
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transgenic mice were not different from other mice and that of Hcrt, the effect of Hcrt-1 and Hcrt-2 was compared in the h
GFPdidnotaltermembraneproperties,wealsorecordedfrom same neurons. Both Hcrt-1 and Hcrt-2 at 50 nM depolarized ttp
age-matched PVT neurons in nontransgenic mice. We did not and excited PVT neurons. Hcrt-2 produced an effect that was ://jn
find any substantive differences in RMP, spike frequency, or as strong as or stronger than Hcrt-1 (Fig. 3E). The mean .p
inputresistancebetweenthethreegroupsofneurons(Table1). depolarizationby50nMHcrt-1andHcrt-2was4.1(cid:4)0.37and hy
s
Allthreegroupsofneuronshadbothactiveandsilentcells, 7.5(cid:4)0.54mV,respectively(n(cid:3)5,P(cid:7)0.01,pairedStudent’s io
insimilarproportions.Wegroupedallthecellsintoactive(n(cid:3) t-test; Fig. 3F). log
111) or silent (n (cid:3) 70) cells. Active cells showed a more The mechanism of Hcrt depolarization was further charac- y.o
substantial H-current and somewhat less SFA (Table 1). terized.ThedepolarizingeffectofHcrt-2(50nM)persistedin rg
the presence of either TTX (1 (cid:1)M; Fig. 3G) or in modified a/
ACSF (0.1 mM Ca2(cid:10)/10 mM Mg2(cid:10); data not shown) that t P
Hcrt excites vGluT2-GFP–positive neurons blocks synaptic transmission. The effect of Hcrt-2 also per- enn
sisted in ACSF containing GABA and glutamate receptor S
renvtGclulaTm2p-GuFsPin–gpotshiteivwehPoVleTcneellurmonosdewearnedreccoomrdpeadreidnwcuitrh- antagonists (BIC, AP5, and CNQX;An (cid:3) 5, data not shown). tate
Thereisnosignificantdifferenceinthedepolarizationinduced U
vGluT2-GFP–negative cells in the same PVT slice. As shown n
in Fig. 3, A and B, and Table 2, (cid:9)71% (54/76) of all the by50nMHcrtamongneuronsinthenormalACSFcondition, iv
TTX condition, or low calcium/high magnesium conditions, o
neurons recorded (whether silent or active) were potently n
andthemeandepolarizationwas7.76(cid:4)0.47(n(cid:3)18),7.5(cid:4) F
depolarized and excited by flow pipette application of Hcrt-2. 0.60(n(cid:3)8),and8.07(cid:4)0.67mV(n(cid:3)10),respectively(P(cid:1) eb
BitievceauasnednvoGdluifTfe2r-eGnFceP–wnaesgafotiuvnedcebleltswreeelnativvGeltuoT2in-cGrFeaPs–epsoisn- 0.5, ANOVA). These results suggest that Hcrt action was a rua
directpostsynapticeffect.Figures3Gand6Gshowathicken- ry
spike frequency, amplitude of depolarization, or percent of 2
ing of the trace after Hcrt-2 application even though synaptic 3
responding cells (Table 2), data are grouped together. Similar potentialswereblocked,suggestinganopeningofsomechan- , 2
results were also found in PVT neurons from nontransgenic 0
nels after Hcrt application. 1
mice (Table 2). As shown in Fig. 3C, 50 nM Hcrt-2 signifi- 3
cantlyincreasedtheaverageactionpotentialfrequencybyover
threefold, from 0.95 (cid:4) 0.19 to 3.15 (cid:4) 0.36 Hz (n (cid:3) 27, P (cid:7) Hcrt depolarization is attributable to two mechanisms:
0.01, ANOVA), and recovered to 1.15 (cid:4) 0.28 Hz after wash- closure of a potassium channel and activation of
outfor5–30min.ThedepolarizingresponsesofPVTtoHcrt-2 nonselective cationic channels
application were found to be concentration dependent at con-
centrations ranging from 10 to 1,000 nM; the EC value was The following experiments were designed to elucidate the
50
25nM(Fig.3D;neuronsthatdidnotrespondtoHcrtwerenot mechanism of the depolarization caused by Hcrt-2. First, we
included). Active and silent cells were also compared in their studied whether the input membrane resistance was changed
response to Hcrt; no differences were found (Table 2). Addi- after Hcrt application. In 8 of 24 neurons depolarized and
tionally,therewasnoobviousdifferenceinlocationwithinthe excited by 50 nM Hcrt-2, the input resistance was increased
PVT of neurons showing different responses to Hcrt (data not after application of Hcrt-2 (Fig. 4A; mean increase, 12.2 (cid:4)
shown). To exclude the possibility that the absence of a 2.7%; P (cid:7) 0.01, paired Student’s t-test). An increase in input
response in the remaining PVT neurons to 50 nM Hcrt-2 was resistance could be caused by the closure of potassium chan-
caused by poor sensitivity to low peptide concentrations, bath nels.Totestthishypothesis,wecomparedtheeffectofHcrt-2
application of 50 nM Hcrt-2 or flow pipette application of 1 on neurons held at a membrane potential of (cid:2)60 mV in two
(cid:1)M Hcrt-2 was used. Similar results were obtained. Bath differentconditionsofexternalpotassiumconcentration([K] ),
o
JNeurophysiol(cid:127)VOL95(cid:127)MARCH2006(cid:127)www.jn.org
PREFRONTAL CORTEX-PVT ACTIVATION BY HYPOCRETIN 1661
To further analyze the contribution of K(cid:10) channels to the
depolarization, we undertook an additional experiment. Volt-
age clamp was used to study the effect of Hcrt on membrane
current.Current-voltageplots(I-Vcurves)wereobtainedinthe
presenceofTTX,CNQX,AP5,andBIC.InnormalACSFwith
[K] at 2.5 mM, a comparison of voltage-clamp ramps in
o
controlandinthepresenceofHcrt(Fig.4,CandE)indicated
a reversal at approximately (cid:2)110 mV of the Hcrt-induced
currentin7of15neuronswhichhadclearinwardcurrentsafter
applicationof50nMHcrt-2.Inthesevencells(Fig.4E,inset),
the mean reversal was (cid:2)110.0 (cid:4) 3.4 mV, very close to the
estimated E . Performing the same protocol in an ACSF with
K
[K] at 15 mM in other PVT neurons indicated a reversal of
o
approximately (cid:2)60 mV of Hcrt-induced currents in five of
eightneurons(Fig.4,DandE)whichhadclearinwardcurrents D
afterHcrt-2application.Inthesefivecells(Fig.4E,inset),the ow
mean reversal was (cid:2)63.0 (cid:4) 2.8 mV, very close to the theo- n
lo
reticalE .Together,theseresultsindicatethatthedepolarizing a
K d
effect of Hcrt in these PVT neurons is mediated by the e
d
reduction of a potassium conductance. fro
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FIG.2. Membrane properties of vGluT2-GFP–positive PVT neurons. A1 iv
and A2: voltage responses to current injection in 2 different neurons. One o
neuron has a clear sag (A2, dot), suggesting the presence of H-current. The n F
other neuron does not have a clear sag (A1, dot). Note presence of time- e
ianmdpeplietunddeenctu(rAre1ntanpdulAse2s) i(n(cid:11)w).arAd3r:ecmtiefiacnaticounr,reanctt-ivvoaltteadgeinrerelastpioonnssheiptoilnar5g5e brua
neurons.Valuesweretakenattheendofpulses(dotsinA1andA2).B:note ry
afterhyperpolarization(AHP).C1:responsetocurrentstepsfromahyperpo- 2
3
larizedlevel,showingstronglowthresholdspike(LTS).C2–C4:tracesshow , 2
thatLTSpersistsinthepresenceofTTX(C2),iseliminatedbynickel(C3), 0
1
andcanrecoverafterwashoutofnickel(C4).D1andD2:voltageresponsesto 3
depolarizing40-and120-pAcurrents,showingclearspikefrequencyadapta-
tion(SFA)(D1)andspikefailure(D2).E1andE2:anothercellshowsonly
modestSFAandnospikefailure.
resultingintwodifferentE s.AsshowninFig.4B1,inthefirst
K
condition ([K] (cid:3) 2.5 mM; estimated E (cid:3) (cid:2)108.3 mV),
o K
Hcrt-2powerfullydepolarizedPVTneurons.Incontrast,when
tested again in the second condition with high K(cid:10) (Fig. 4B2;
[K] (cid:3) 15 mM; estimated E (cid:3) (cid:2)60.5 mV), application of FIG.3. Hypocretins excite GFP-positive PVT neurons. A and B: Hcrt-2
Hcrot-2hadnocleareffect.TheKdepolarizationby50nMHcrt-2 depolarizesandexcitesasilent[A,restingmembranepotential(RMP),(cid:2)59.2
in these two conditions was 9.8 (cid:4) 0.55 and 1.4 (cid:4) 0.8 mV, mnMV)]oonrsapnikaectfirveequ(Ben,cRyMofPP,V(cid:2)T58(.*8*PmV(cid:7))0n.0eu1r,onn(cid:3).C2:7m).eDan:deofsfee–ctreosfpoHncsret-c2u(r5v0e
respectively (n (cid:3) 5, P (cid:7) 0.01, unpaired Student’s t-test; Fig. fortheeffectofHcrt-2ondepolarizationinPVTneurons(n(cid:3)5at5nM,4
4B3). The effect of Hcrt on membrane depolarization showed at10nM,4at20nM,15at50nM,4at100nM,and7at200nM).E:response
little desensitization. In normal conditions, the depolarization toHcrt-1andHcrt-2(bothat50nM)ofasingleneuron(RMP,(cid:2)59.3mV).F:
by Hcrt-2 (50 nM) was 7.8 (cid:4) 0.3 and 7.6 (cid:4) 0.4 mV (n (cid:3) 3) comparisonofthedepolarizationinducedbyHcrt-1andHcrt-2(appliedat50
nM; **P (cid:7) 0.01, n (cid:3) 5). G: in the presence of TTX (1 (cid:1)M), Hcrt-2
for the first and second application of Hcrt with a 15-min
depolarized the membrane potential and caused a thickening of the trace,
interstimulus interval, respectively. probablybecauseofchannelopenings(RMP,(cid:2)59.2mV).
JNeurophysiol(cid:127)VOL95(cid:127)MARCH2006(cid:127)www.jn.org
1662 H. HUANG, P. GHOSH, AND A. N. VAN DEN POL
TABLE 2. Responses of different cell groups to Hcrt-2
IncreaseofSFby DepolarizationbyHcrt-2,
HcrtResponse NoHcrtResponse Hcrt-2,50nM n 50nM n
vGluT2-GFP(cid:10)cells 74%(34/46) 26%(12/46) 3.37-fold 20 7.77(cid:4)0.64mV 11
vGluT2-GFP(cid:2)cells 67%(20/30) 33%(10/30) 3.12-fold 7 7.71(cid:4)0.61mV 7
Cellsinnontransgenicmice 75%(25/33) 25%(8/33) 3.40-fold 11 7.94(cid:4)0.58mV 8
Silentcells 74%(34/46) 26%(12/46)
Activecells 71%(45/63) 29%(18/63)
Valuesaremeans(cid:4)SD.Numbersinparenthesesrefertothenumberofcellswiththatproperty—thetotalnumberofcellstested.SF,spikefrequency.
In addition to an increase in input resistance, of the 24 out the anteroposterior extent of the PVT. Figure 6B is an
neurons depolarized and excited by 50 nM Hcrt-2 described exampleshowingthelabelingofneuronsinthePVT.Labeling
above, 9 other neurons showed a decrease (Fig. 5A; mean was mainly on one side, ipsilateral to the side of tracer
decrease,11.9(cid:4)2.4%;P(cid:7)0.01,pairedStudent’st-test),and injection (Fig. 6B). Within the thalamus, retrogradely labeled D
the remaining 7 neurons showed no clear change (Fig. 5B; neuronswerefoundinthePVTandalsointhemediodorsaland ow
meanincrease,1.0(cid:4)1.3%;P(cid:1)0.5,pairedStudent’st-test)in reuniens nucleus of thalamus. In contrast, few neurons of the n
lo
inputresistance.Furthermore,in8ofthe15neuronsdescribed central medial thalamus nucleus showed labeling. Figure 6, C a
d
in the paragraph above that displayed a clear inward current and D, shows the labeling of the vGluT2-GFP–positive neu- e
d
afterapplicationof50nMHcrt-2involtageclamp,thereversal rons and red tracer labeled neurons in the same micrograph. fro
of the Hcrt-induced current was (cid:2)38.0 (cid:4) 3.1 mV in one-half Figure6,C–E,showsthatsomevGluT2-GFP–positiveneurons m
(4)ofthem(Fig.5,C1andC2),andintheremainingone-half wereretrogradelylabeled(indicatedbythearrows),indicating h
n(4o)tocfotnhveerngeeuroovnesr,tthheeIe-nVtilrienevsorlteamgeainraendgeeittheesrtepdar(aFlilgel. o5r, dDi1d tghlauttatmheasteerPgiVc.TBneecuaruosnesopurrojCecTtiBngintjoectthieonmsPitFeCs ianrethperombPabFlCy ttp://jn
and D2). weresmall,thepercentofretrogradelylabeledPVTneuronsis .ph
TheaboveresultthatHcrt-inducedcurrentsreversedat(cid:2)38 probably an underestimate of the total number of cells that ys
io
mV is consistent with nonselective cationic channels as the project from the PVT to the mPFC. lo
basis for the currents and suggest that activation of nonselec- ToinvestigatewhethertheGFP-expressingneuronsproject- gy
tive cationic channels contribute to the depolarizing effect of ingtomPFCinPVTwereHcrtrespondersornot,werecorded .o
rg
Hincwrta.rHdocwuervreenr,tanwoothueldrmbeechtahneismelethctartomgeignhictcNonat(cid:10)ri/bCuat2e(cid:10)toethxe- fbreoemn rinedjecPtVedTinnetourtohnesmrePtrFoCgriandevlGylluaTbe2l-eGdFwPitmhiCceT.Bthathad at P/
changer,whichhasasimilarreversalpotential(Burdakovetal. The retrogradely labeled neurons were either quiescent or e
n
2003; Ehara et al. 1989). The inward current mediated by the fired regular spontaneous action potentials. The RMP was n
Na(cid:10)/Ca2(cid:10) exchanger is dependent on the availability of cyto- (cid:2)60.0(cid:4)0.6mV(n(cid:3)60).Theinputmembraneresistancewas Sta
solic Ca2(cid:10). Cytosolic Ca2(cid:10) comes from the extracellular en- 358(cid:4)10M(cid:6)(n(cid:3)37).Forty-sevenpercent(18of38)ofthe te
U
vironment or from intracellular stores. We therefore repeated neurons had clear H-currents. Most neurons (8 of 10) showed n
the above experiments with modified (0 Ca2(cid:10)/4 mM Mg2(cid:10)) clear SFA or spike failure with greater current injections. All iv
o
ACSF and recorded with a pipette solution containing 10 mM the neurons studied showed inward rectification and LTS. n
BAPTA, a high-affinity Ca2(cid:10) chelator. In 16 neurons that had ApplicationofHcrt-2(50nM)depolarizedandexcited(cid:9)76% Fe
b
clear inward currents after application of 50 nM Hcrt-2 in (25of33)oftheretrogradelylabeledneurons(Fig.6F).After ru
nominal zero calcium ACSF, we still found that in 6 neurons Hcrt application, the mean frequency of spontaneous action a
the Hcrt-induced currents reversed at (cid:2)35.8 (cid:4) 2.38 mV; in 7 potentials increased by about threefold, from 0.93 (cid:4) 0.24 to ry 2
nreemuraoinnsintghe3cnuerureronntss,rethveerIs-eVdlaitne(cid:2)s1r0e9m.2ai(cid:4)ned3.p9a3rmallVel;oanrddiidnnthoet r2e.9tr4og(cid:4)rad0e.l5y5laHbzel(end(cid:3)GF2P0(cid:10), Pneu(cid:7)ro0n.s0,19, oAfNtOheVmAw).eIrne 1d1epColTaBr- 3, 20
1
converge (data not shown). These results are consistent with ized and excited by Hcrt-2 (from 0.96 (cid:4) 0.26 to 2.73 (cid:4) 0.49 3
the previous findings and suggest that the Hcrt-induced cur- Hz,P(cid:7)0.01,ANOVA).Inthepresenceof1(cid:1)MTTX,Hcrt-2
rents that reversed around (cid:2)38 mV are likely caused by the (50 nM) still depolarized neurons that had been retrogradely
activation of nonselective cationic channels and not by the labeledbyCTB(Fig.6G),suggestingagaintheinvolvementof
Na(cid:10)/Ca2(cid:10) exchanger. a postsynaptic action. The mean depolarization by Hcrt-2 was
Different PVT neurons showed different mechanisms of 8.7 (cid:4) 1.2 mV (n (cid:3) 5). The changes of input membrane
response. We found no obvious anatomical differences in resistance were also studied in these retrogradely labeled neu-
different groups of responding neurons or any anatomical rons. In 16 neurons that were depolarized and excited by
correlation with the existence of H-current or spontaneous Hcrt-2(50nM),theinputresistancewasincreasedin5neurons
activity (data not shown). (mean increase, 16.4 (cid:4) 3.9%; P (cid:7) 0.01, paired Student’s
t-test); decreased in 4 neurons (mean decrease, 15.2 (cid:4) 4.7%;
P (cid:7) 0.01, paired Student’s t-test); and not significantly
Cortically projecting vGluT2-GFP–positive PVT neurons are
changed in 7 neurons (mean decrease, 1.0 (cid:4) 1.8%; P (cid:1) 0.5,
excited by Hcrt
paired Student’s t-test), implying that multiple mechanisms
Retrograde tracer CTB injections into mPFC typically in- were also involved in these neurons. Together, these results
volved both the prelimbic and infralimbic areas of the PFC indicatethatHcrthasdirectexcitatoryactionsonthecortically
(Fig.6A).Thiscorticalinjectionoftracerlabeledcellsthrough- projecting PVT neurons.
JNeurophysiol(cid:127)VOL95(cid:127)MARCH2006(cid:127)www.jn.org
PREFRONTAL CORTEX-PVT ACTIVATION BY HYPOCRETIN 1663
and its agonists and GABA agonist were applied through a
flowpipe.Inalltheneuronstested,glutamate(50(cid:1)M,n(cid:3)5),
AMPA (30 (cid:1)M, n (cid:3) 6), and NMDA (50 (cid:1)M, n (cid:3) 7)
depolarizedtheneuronsandevokedgroupsofspikesincurrent
clamp (data not shown). In voltage clamp ((cid:2)60 mV holding
potential), inward currents were generated, and the inward
currents evoked by glutamate, AMPA, and NMDA were
13.4 (cid:4) 1.9, 137.5 (cid:4) 27.3, and 44.4 (cid:4) 4.7 pA, respectively.
Muscimol (20 (cid:1)M), a GABA receptor agonist, attenuated
A
spikefrequencyandhyperpolarizedthemembranepotentialby
28.1(cid:4)1.0mV(n(cid:3)6).ThusglutamateandGABAreceptors
are expressed in PVT neurons.
Bothexcitatoryandinhibitorysynapticactivitywasfoundin
PVT. Using a recording pipette solution with KMeSO with
theGABA antagonistBIC(30(cid:1)M)inthebath,sponta4neous Do
excitatorypAostsynapticcurrents(EPSCs)wereidentified((cid:2)60 wn
mVholdingpotential).TheseEPSCswerecompletelyblocked loa
byglutamateionotropicantagonistsAP5(50(cid:1)M)andCNQX de
7(1A0).(cid:1)InMt)haenpdrerseecnocveeroefdAaPft5eranandtaCgNoQniXstiwnatshheobuatt(hn,s(cid:3)po6n;taFnieg-. d from
ous inhibitory postsynaptic currents (IPSCs) were recorded h
using a recording pipette solution containing KCl ((cid:2)60 mV ttp
holding potential). These currents were blocked by BIC and ://jn
recoveredafterBICwashout(Fig.7B).PVTreceivedarobust .p
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FIG.4. HypocretinexcitesPVTbyblockingaK(cid:10)current.A:comparisonof
hyperpolarizingpulsesbefore(1)andduring(2)effectofHcrt-2.RMP,(cid:2)59.3
mV.Bottominset:enlargementofpulsesshowingincreaseininputmembrane
resistanceinthepresenceofHcrt-2.B1andB2:depolarizingeffectofHcrt-2(B1)
issuppressed(B2)afterchanging[K] from2.5to15mMwhenneuronsareheld
o
at (cid:2)60 mV. B3: comparison of depolarization induced by Hcrt-2 in external
solutionwith[K] of2.5or15mM(**P(cid:7)0.01,n(cid:3)5).C:voltage-clampramps
o
intheabsence(b)orpresence(a)ofHcrt-2with[K] of2.5mM.D:voltage-clamp
o
ramps in the absence (d) or presence (c) of Hcrt-2 with [K] of 15 mM. E:
o FIG.5. HypocretinexcitesPVTthroughactivationofnonselectivecationic
subtractionofcurvesfromC(aandb)andD(candd).Inset:reversalpotentials
currents.AandB:comparisonofhyperpolarizingpulsesbefore(1)andduring
forbothconditionswithrespecttoNernstrelationship. (2) effect of Hcrt-2 in 2 neurons. RMP, (cid:2)58.9 mV in A; (cid:2)59.2 mV in B.
Bottominset:enlargementofpulsesshowingdecrease(A)orlackofchange
Hcrt has little effect on glutamate or GABA synaptic
(B) in input membrane resistance in the presence of Hcrt-2. C1 and D1:
currents in PVT voltage-clamprampsinthepresence(a)andabsence(b)ofHcrtin2neurons.
C2andD2:subtractionofcurves(aandb)fromC1andD1,respectively.C1
Prior to studies of synaptic input, to ensure that PVT ex- and C2: Hcrt-2–induced current reversed at (cid:2)38 mV. D1 and D2: Hcrt-2–
pressed ionotropic glutamate and GABA receptors, glutamate inducedcurrentremainsparallel.
JNeurophysiol(cid:127)VOL95(cid:127)MARCH2006(cid:127)www.jn.org
1664 H. HUANG, P. GHOSH, AND A. N. VAN DEN POL
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FIG.6. DirecteffectofhypocretinonvGluT2-GFP–positiveneuronsinPVTthatprojecttoprefrontalcortex.A:representativeinjectionsite.Choleratoxin
subunitB(CTB)wasinjectedinthemPFC.IL,infralimbicarea;PL,prelimbicarea.Arrowsshowmidlineofbrain.B:PVTwasselectivelylabeled,ipsilateral
toinjectionside.Mediodorsal(MD),andreuniensnucleus(RE)werealsolabeled,whereasfewcentralmedial(CM)nucleuswerelabeled.CandD:GFP-positive
neurons(GFP(cid:10),C)andretrogradelylabeledneurons(CTB(cid:10),D)inasamelayerofPVT.E:bothGFP(cid:10)andCTB(cid:10)neuronsareinPVT(boxedareainCand
D).Double-labeledneuronsshowayellowcolor.ArrowsinC–Eshow3neuronsthataredouble-labeled.ArrowheadsinB–Dshowmiddleofventricle.F:
retrogradelylabeledvGluT2-GFP–positiveneuronisdepolarizedandexcitedbyHcrt-2(50nM).RMP,(cid:2)58.9mV.G:Hcrt-2(50nM)depolarizedaretrogradely
labeledvGluT2-GFP–positiveneuroninthepresenceofTTX(1(cid:1)M).RMP,(cid:2)59.7mV.Scalebars:A,200(cid:1)m;B,100(cid:1)m;CandD,50(cid:1)m;E,125(cid:1)m.
JNeurophysiol(cid:127)VOL95(cid:127)MARCH2006(cid:127)www.jn.org