indirect projections from the suprachiasmatic nucleus to major arousal-promoting cell groups in rat

19 Pages·2004·0.54 MB·English

Neuroscience130(2005)165–183
INDIRECT PROJECTIONS FROM THE SUPRACHIASMATIC NUCLEUS
TO MAJOR AROUSAL-PROMOTING CELL GROUPS IN RAT:
IMPLICATIONS FOR THE CIRCADIAN CONTROL OF BEHAVIOURAL
STATE
S.DEURVEILHERANDK.SEMBA* Thecircadianclockhousedinthesuprachiasmaticnucleus
(SCN) of the hypothalamus governs daily rhythms of
DepartmentofAnatomyandNeurobiology,FacultyofMedicine,Dal-
housieUniversity,5850CollegeStreet,Halifax,NovaScotia,Canada sleep–wakingandotherbehavioralandphysiologicalfunc-
B3H1X5 tions (Rusak and Zucker, 1979; Moore and Leak, 2001).
SCNlesionsinrodentsabolishdailysleep–wakerhythms;
interestingly, however, the amounts of sleep or wakeful-
Abstract—The circadian clock housed in the suprachias-
nessarenotaffected(Ibukaetal.,1977;WurtsandEdgar,
matic nucleus (SCN) controls various circadian rhythms in-
cludingdailysleep–wakecycles.Usingdualtract-tracing,we 2000).ThesefindingssuggestthattheSCNisnotrespon-
recently showed that the medial preoptic area (MPA), sub- sibleformaintainingbehavioralstates,butinfluencestheir
paraventricular zone (SPVZ) and dorsomedial hypothalamic timinginacircadianmanner.Behavioralstatesare,infact,
nucleus(DMH)arewellpositionedtorelaySCNoutputtotwo controlledbyaneuralsystemthatnetworksacrosswidely
key sleep-promoting nuclei, namely, the ventrolateral and distributed brain regions (Steriade and McCarley, 1990;
medianpreopticnuclei.Thepresentstudyexaminedthepos-
LydicandBaghdoyan,1999).Someofthecircadianinflu-
sibility that these three nuclei may link the SCN with wake-
ence on the sleep/wake-regulatory system is likely to be
regulatoryneuronalgroups.Biotinylateddextran-aminewith
mediated by humoral factors (LeSauter and Silver, 1998;
orwithoutcholeratoxinBsubunitwasinjectedintoselected
maintargetsofSCNefferents;theretrogradelabelinginthe Krameretal.,2001;Chengetal.,2002).However,“hard-
SCN was previously analyzed. Here, anterograde labeling wired” synaptic pathways certainly play a role as well
was analyzed in immunohistochemically identified cholin- (Swanson, 1987; Buijs and Kalsbeek, 2001; Pace-Schott
ergic, orexin/hypocretin-containing and aminergic cell and Hobson, 2002), and little is known about these path-
groups. Tracer injections into the MPA, SPVZ and DMH re- ways.
sultedinmoderatetodenseanterogradelabelingofvaricose Following the general organizational principle of the
fibersintheorexinfieldandthetuberomammillarynucleus.
hypothalamus(Swanson,1987),theSCNdoesnotproject
The locus coeruleus, particularly the dendritic field, con-
abundantlybeyondthehypothalamus(Wattsetal.,1987).
tained moderate anterograde labeling from the MPA and
No region in the sleep–wake regulatory system receives
DMH.Theventraltegmentalarea,dorsalraphenucleus,and
laterodorsaltegmentalnucleusallshowedmoderateantero- directprojectionsfromtheSCN,withtheexceptionofthe
grade labeling from the DMH. The substantia innominata medial preoptic area (MPA), which receives strong direct
showedmoderateanterogradelabelingfromtheMPA.These SCN projections (Watts et al., 1987; Leak and Moore,
results suggest that the MPA, SPVZ and DMH are possible 2001),aswellastheventrolateralpreopticnucleus(Novak
relay nuclei for indirect SCN projections not only to sleep- andNunez,2000;Chouetal.,2002)andtheareacontain-
promoting preoptic nuclei as previously shown, but also to
ing orexin (also known as hypocretin)-containing neurons
wake-regulatorycellgroupsthroughoutthebrain.Intheab-
in the posterior hypothalamus (Abrahamson et al., 2000),
senceofmajordirectSCNprojectionstomostofthesesleep/
whichbothreceivesparsedirectSCNprojections.Indirect
wake-regulatoryregions,indirectneuronalpathwaysproba-
pathwaysarecertainlyconceivable,however,andwehave
blyplayanimportantroleinthecircadiancontrolofsleep–
wakecyclesandotherphysiologicalfunctions.©2004IBRO. recently shown, using a dual tract-tracing method, that
PublishedbyElsevierLtd.Allrightsreserved. several known targets of direct SCN projections are well
placed to relay SCN output to the ventrolateral (Deurveil-
Key words: dual tract-tracing, immunohistochemistry, cho- her et al., 2002) and median preoptic nuclei (Deurveilher
linergic,orexin/hypocretin,aminergic.
and Semba, 2003), two key structures involved in the
generationofsleep(McGintyandSzymusiak,2001;Saper
*Correspondingauthor.Tel:(cid:1)1-902-494-2008;fax:(cid:1)1-902-494-1212.
E-mailaddress:[email protected](K.Semba). et al., 2001). These potential relay nuclei are the MPA,
Abbreviations: A, anterior; BDA, biotinylated dextran-amine; CTB, subparaventricular zone (SPVZ), and dorsomedial hypo-
cholera toxin B subunit; DAB, diaminobenzidine; DMH, dorsomedial thalamic nucleus (DMH). The synaptic connections that
hypothalamicnucleus;HDB,horizontallimbofthediagonalbandof
formtherelaysineachofthesenucleiremaintobeinves-
Broca; HDC, histidine decarboxylase; ir, immunoreactive; LD, light–
dark; MPA, medial preoptic area; MS-VDB, medial septum-vertical tigated. However, in the absence of major direct projec-
limbofthediagonalbandcomplex;NPY,neuropeptideY;P,posterior; tionstothesleep–wakeregulatorysystem,thesepotential
PH, posterior hypothalamic area; REM, rapid eye-movement; SCN,
indirect pathways may play an important role in circadian
suprachiasmatic nucleus; SPVZ, subparaventricular zone; TH, ty-
rosinehydroxylase;VAChT,vesicularacetylcholinetransporter. regulationofsleep.
0306-4522/05$30.00(cid:1)0.00©2004IBRO.PublishedbyElsevierLtd.Allrightsreserved.
doi:10.1016/j.neuroscience.2004.08.030
165
166 S.DeurveilherandK.Semba/Neuroscience130(2005)165–183
Inthepresentstudy,usingthesamedualtract-tracing nucleus;andratanti-cholineacetyltransferase(1:50;Boehringer,
approach, we investigated whether the previously identi- Laval, Quebec, Canada) or rabbit anti-vesicular acetylcholine
fied potential relay nuclei to the ventrolateral and median transporter(VAChT;1:15,000;ChemiconInternational)tovisual-
izecholinergicneuronsinthebasalforebrain[themedialseptum–
preopticnucleimayalsorelaySCNoutputtomajorarous-
verticallimbofthediagonalbandcomplex(MS-VDB),horizontal
al/wake-promoting neuronal groups, including cholinergic
limbofthediagonalbandofBroca(HDB),magnocellularpreoptic
neurons in the basal forebrain and mesopontine tegmen- nucleus,andsubstantiainnominata]andinthemesopontineteg-
tum;orexin-containingneuronsintheposteriorhypothala- mentum (the pedunculopontine and laterodorsal tegmental
mus;andaminergicneuronsinthetuberomammillarynu- nuclei).
cleus, ventral tegmental area, substantia nigra pars com- In addition to the sections processed for double labeling as
above,asecondseriesofsectionswasreactedtovisualizeBDA
pacta, dorsal raphe nucleus and locus coeruleus. All of
singlyforquantitativeanalysisofanterogradelabelingintheareas
these neuronal populations are thought to play important
mentionedabove.
rolesinthecontrolofcorticalandbehavioralarousal,and
somearealsoimplicatedinthemechanismsofrapideye- Dataanalysis
movement (REM) sleep (see above). The density of an-
The density of anterograde labeling in the wake-related cell
terogradely labeled varicose fibers was analyzed in each
groupswasassessedusingeithertwoorthreesectionsforeach
of these wake-promoting structures. Preliminary results cellgroupatthefollowinganterior(A)toposterior(P)levels(mm
have been reported in abstract form (Deurveilher et al., to bregma) according to the brain atlas by Paxinos and Watson
2001a,b,c). (1998): MS-VDB (A 0.7 and A 0.2); HDB (A 0.1 and P 0.3);
magnocellular preoptic nucleus (P 0.3 and P 0.8); substantia
EXPERIMENTAL PROCEDURES innominata(P1.3andP1.6);orexincellgroup(P2.8andP3.14);
tuberomammillary nucleus (dorsal division: P 3.8 and P 4.16;
Asafollowuptotwopreviousstudiesfromourlaboratory(Deur- ventraldivision:P4.16andP4.3);ventraltegmentalarea(P5.3
veilheretal.,2002;DeurveilherandSemba,2003),weperformed andP5.6)andsubstantianigraparscompacta(P5.8andP6.04);
additional histological processing of brain sections from the ani- dorsalraphenucleus(P7.8,P8.2andP8.8);pedunculopontine
malsusedinthesepreviousstudies.Thedetailsofanimalsused, (P7.64andP8.3)andlaterodorsaltegmentalnuclei(P8.8andP
tracerinjections,perfusion,andimmunohistochemicalprocedures 9.16);andlocuscoeruleus(dendriticregion:P9.16andP9.68;
were described in Deurveilher et al. (2002). Briefly, adult male cell body region: P 9.68 and P 10.64). Note that the dendritic
Wistarratswereinjected,underanesthesia(60mg/kgketamine, regionofthelocuscoeruleus,whichwasdelineatedbyadense
3.2 mg/kg xylazine, and 0.6 mg/kg acepromazine, i.m.), with a plexus of TH-immunoreactive (-ir) dendrites, overlaps with Bar-
mixture of biotinylated dextran-amine (BDA; 10% in saline) and rington’s nucleus at P 9.16 (Rizvi et al., 1994; Steininger et al.,
choleratoxinsubunitB(CTB;0.25or0.5%),orBDAalone(10%), 2001).Inadditiontotheaboveregions,thedensityofanterograde
into the MPA (n(cid:2)10), SPVZ (n(cid:2)11), DMH (n(cid:2)8), or posterior labelingwasevaluatedinregionsofthemesencephalicreticular
hypothalamic area (PH; n(cid:2)7). Postinjection survival periods formation(P6.72andP7.04),involvedinarousal,andthepontine
ranged from 8 to 11 days (mostly either 9 or 11 days) after reticular formation (P 8.8 and P 9.3), involved in REM sleep
co-injections of BDA and CTB, and from 4 to 10 days (mostly generation(SteriadeandMcCarley,1990).
either 5 or 6 days) after single injections of BDA. No obvious Asinourpreviousstudies(Deurveilheretal.,2002;Deurveil-
differences were noted among the cases with different survival herandSemba,2003),anterogradelabelingwasanalyzedusing
periods. All experiments were performed in compliance with the sections stained only for BDA, because the additional immuno-
guidelinesestablishedbytheCanadianCouncilonAnimalCare staining often interfered with the evaluation of BDA-labeled ele-
and by the Dalhousie University Committee on Laboratory Ani- ments. Transmitter-specific cell groups in BDA-labeled sections
mals. All efforts were made to minimize the number of animals were identified by comparing with adjacent sections double-
usedandtheirsuffering. stainedforBDAandarelevanttransmittermarker.Thedensityof
TheinjectionsitesandtheretrogradelabelingintheSCNin anterograde labeling was assessed within a region that closely
relationtothevasopressin-richshell,andneuropeptideY(NPY)- outlinedtheentireclusterofimmunolabeledcellbodiesandprox-
rich core regions have been reported previously in detail (Deur- imaldendritesforallthecellgroupsexceptthelocuscoeruleus,
veilheretal.,2002).Forconvenience,someoftheseresultsare basalforebrainregions,orexinfield,andmesencephalicandpon-
includedinthepresentstudy,withreferencetotheoriginalsource. tinereticularformation.Forthelocuscoeruleus,dendriticandcell
ToexamineanterogradeBDAlabelinginthewake-regulatory bodyregionswereanalyzedseparatelyasthedensityofantero-
structures,aseriesof40-(cid:3)mcoronalsectionsfromeachcasewas grade labeling appeared denser in the dendritic, than the cell
reacted with a standard avidin–biotin–horseradish peroxidase body, region. For the three basal forebrain regions, the orexin
complex method, using diaminobenzidine (DAB) and nickel am- field,andthemesencephalicandpontinereticularformation,den-
moniumsulfatetoproduceablack–purplereactionproduct(Deur- sityanalysisboxeswereused.FortheMS-VDB,abox(0.4mmin
veilheretal.,2002).Thesesectionswerethenreactedimmunohis- width(cid:4)1.5mminheightatA0.7;0.4mm(cid:4)2.5mmatA0.2)was
tochemically with a peroxidase anti-peroxidase method and DAB placedsothatitsmedialborderwasalignedwiththemidlineofthe
without nickel to produce a brown reaction product. The following brainsection.FortheHDB,abox(1mm(cid:4)0.4mm)waspositioned
primaryantibodieswereused:rabbitanti-preprohypocretinantibody tangential to the ventral surface of the brain. For the substantia
(1:1000;ChemiconInternational,Temecula,CA,USA)tovisualize innominata,abox(1mm(cid:4)0.6mm)wasplacedsubjacenttothe
orexin/hypocretin-containingneuronsintheposteriorhypothalamus; globus pallidus; no box was required for the magnocellular pre-
rabbitanti-histidinedecarboxylase[HDC;1:1600;kindlyprovidedby opticnucleus,whichcanbedelineatedeasilyinsectionssingle-
Dr.N.Inagaki,OsakaUniversity,Osaka,Japan]tovisualizehista- stainedforBDAonthebasisofafaintnon-specificstainingofits
minergic neurons in the tuberomammillary nucleus; rabbit anti-ty- magnocellular neurons. For the orexin cell group, a box
rosine hydroxylase (TH; 1:6000; Pel-Freez, Rogers, AR, USA) to (1 mm(cid:4)0.5 mm) was placed so that the midpoint of its ventral
visualize dopaminergic neurons in the ventral tegmental area and segmentwascenteredatthefornix,thusencompassingtheperi-
substantianigraparscompacta,andnoradrenergicneuronsinthe fornical area where orexin neurons are most concentrated; the
locuscoeruleus;rabbitanti-serotonin(1:20,000;Diasorin,Stillwater, analysis box was then divided into two halves to analyze the
MN,USA)tovisualizeserotoninergicneuronsinthedorsalraphe medial and lateral portions separately, as anterograde labeling
S.DeurveilherandK.Semba/Neuroscience130(2005)165–183 167
appeareddenserinthemedial,thanthelateral,portion.Forthe
mesencephalic reticular formation, a box (1.5 mm(cid:4)1 mm) was
placed over the deep mesencephalic nucleus. For the pontine
reticular formation, a box (1.5 mm(cid:4)1 mm) was placed over the
ventral part of either the rostral or caudal pontine reticular field
where microinjection of cholinergic agonist can increase the
amountofREMsleepinrat(Deurveilheretal.,1997).Duetothe
predominantlyipsilateralnatureofallthestaining,dataanalyses
wereconductedipsilaterallytotheinjectionsite.
Thedensityofvaricosefibersineachwake-regulatorycellgroup
wasassessedbyusingthesamedensityscaletemplateasinour
previousstudy(DeurveilherandSemba,2003):absent((cid:5)),low((cid:1)),
moderate((cid:1)(cid:1)),anddense((cid:1)(cid:1)(cid:1);Fig.1).Thedensitiesfromeither
twoorthreesectionsineachanimalwereaveragedforeachstruc-
turebecause,ingeneral,therewerenoobviousrostrocaudaldiffer-
encesindensity.Forconsistency,allanalyseswereconductedby
the same examiner (S.D.). Digitized images of selected sections
were captured with an AxioCam video camera (Zeiss), and the
brightness,contrastandsharpnesswereadjustedforpresentation.
Table1liststheabbreviationsusedinFigs.2–4andTables2–5.
RESULTS
Thepresentresultswerederivedfromthe36ratsdescribed
in Deurveilher et al. (2002) and Deurveilher and Semba
(2003).Twenty-sevenratsreceivedco-injectionsofBDAand
CTB,andninereceivedsingleinjectionsofBDA,intooneof
thethreemaintargetnucleioftheSCNprojections,namely
the MPA, SPVZ, and DMH, as well as into the PH. Tables
2–5showtheresultsofallthesecases,includingtheinjection
sites, and the quantitative analysis of retrograde labeling in
theSCNprimarilybasedonCTBlabeling,asadaptedfrom
oneofourpreviousstudies(Deurveilheretal.,2002).The36
caseswerecategorizedintofourgroupsbasedontheinjec-
tionsite:MPA(n(cid:2)10);SPVZ(n(cid:2)11);DMH(n(cid:2)8);andPH
(n(cid:2)7;Deurveilheretal.,2002).
Candidates for relay nuclei that link the SCN with
wake-regulatory neuronal groups were identified by the
presence of both strong retrograde labeling in the SCN,
and strong anterograde labeling in the wake-regulatory
groups. Quantitative criteria for the strong labeling were
established as follows, based on the results of the 36
cases (Deurveilher et al., 2001a,b,c, 2002; Deurveilher
and Semba, 2003), as well as previous studies by others
on the efferent projections of the SCN (Watts and Swan-
son, 1987; Leak and Moore, 2001): (cid:6)20 CTB-labeled or
(cid:6)5 BDA-labeled neurons in the SCN per section, and at
least moderate BDA-labeled varicose fibers in a given
wake-regulatorycellgroup,forcaseswithmediumtolarge
injection sites; (cid:6)5 CTB-labeled or (cid:6)2 BDA-labeled neu-
rons in the SCN per section, and at least low-moderate
BDA-labeledvaricosefibersinagivenwake-regulatorycell
group, for cases with small injection sites. The results
reported below highlight the cases with the highest num- Fig.1. Thedensity-scaletemplateusedforassessingthedensityof
anterograde labeling. Density: absent ((cid:5)), low ((cid:1)), moderate ((cid:1)(cid:1))
bersofretrogradelylabeledneuronsintheSCN;thecases anddense((cid:1)(cid:1)(cid:1)).Semi-levelswerealsousedasrequired.
that yielded few or no retrogradely labeled neurons are
mentionedassupplementarydata.
containingneurons,andthecore,asdefinedbythepres-
ence of NPY-containing fibers (Deurveilher et al., 2002).
TheMPA
The retrograde CTB labeling following MPA injections in-
The retrograde labeling in the SCN was examined with dicated that the SCN projections to the MPA arose pre-
respect to the two anatomical compartments of the SCN: dominantly from the shell, with a small contribution from
the shell, as defined by the presence of vasopressin- the core (Table 2). Strong retrograde labeling was also
168 S.DeurveilherandK.Semba/Neuroscience130(2005)165–183
Table1.AnatomicalabbreviationsusedinfiguresandTables2–5 retrogradelabelingintheSCN,buttheanterogradelabel-
ingdatawereconsistentwiththeabove.Specifically,one
3V Thirdventricle injection in the rostral MPA (504) resulted in greater an-
4V Fourthventricle terograde labeling in the substantia innominata than did
AH Anteriorhypothalamicarea
another,caudalMPAinjection(453).Athirdinjectionwhich
AM Anteromedialthalamicnucleus
was centered dorsally in the MPA yielded anterograde
Bar Barrington’snucleus
labeling in the MS-VDB and, to a lesser degree, in the
BST Bednucleusofthestriaterminalis
CM Centralmedialthalamicnucleus morecaudalbasalforebraincholinergicregions(406).Fi-
DA Dorsalhypothalamicarea nally, a small injection that was centered in the bed nu-
DMH Dorsomedialhypothalamicnucleus cleusofthestriaterminalis,thuslargelymissingtheMPA,
DRN Dorsalraphenucleus yielded little anterograde labeling in any basal forebrain
fx Fornix region(407).
GP Globuspallidus
In the brainstem, only sparse or no anterogradely la-
LCc Locuscoeruleus,coreornuclearregion
beled fibers were seen in the pedunculopontine or the
LCd Locuscoeruleus,dendriticregion
laterodorsaltegmentalnucleusinalltheMPAcases.
LDT Laterodorsaltegmentalnucleus
LS Lateralseptum
Anterograde labeling in the orexin cell region. In all
me5 Mesencephalictrigeminaltract
thesixcaseswithstrongretrogradelabelingintheSCN(472,
MnPO Medianpreopticnucleus
473, 481, 482, 501 and 503), BDA-labeled varicose fibers
MPA Medialpreopticarea
opt Optictract weredenserinthemedial(moderate-densetodense),than
ox Opticchiasm thelateral(lowtomoderate),partoftheorexincellfield(Table
PeF Perifornicalarea 2). Many varicose fibers were seen near the orexin-ir cell
PH Posteriorhypothalamicarea bodiesorproximaldendrites(Fig.2C).
PVH Paraventricularhypothalamicnucleus
Of the two MPA injections that resulted in only a few
RCh Retrochiasmaticarea
retrogradely labeled neurons in the SCN, one injection
Re Reuniensthalamicnucleus
(504) produced greater labeling in the medial, than the
SCN Suprachiasmaticnucleus
scp Superiorcerebellarpeduncle lateral, part of the orexin field, consistent with the prefer-
SI Substantiainnominata entialdistributionofterminationdescribedabove;thesec-
SP Subparafascicularthalamicnucleus ond, caudal injection (453) yielded virtually no labeling in
SPVZ Subparaventricularzone thisregion.
TMNd Tuberomammillarynucleus,dorsalpart
VLPO Ventrolateralpreopticnucleus Anterogradelabelingintheaminergiccellregions. In
VMH Ventromedialhypothalamicnucleus the six cases of MPA injections yielding strong retrograde
VTA Ventraltegmentalarea labeling in the SCN (472, 473, 481, 482, 501 and 503),
moderateanterogradelabelingwasseeninthedorsaltube-
romammillarynucleus,andslightlylighterlabelingintheven-
observedintheventrallateralseptum,bednucleusofthe
traltuberomammillarynucleus(Table2).Inthedorsaltube-
stria terminalis, ventromedial hypothalamic and arcuate
romammillarynucleus,manylabeledvaricositieswerefound
nuclei,andtheamygdala(Deurveilheretal.,2002).
nearHDC-irperikarya(Fig.2D).Theanterogradelabelingin
The anterograde BDA labeling was analyzed in six
the ventral tegmental area and substantia nigra pars com-
casesforbothforebrainandbrainstem,andinfourcases
pactawassparsetolow.Thedorsalraphenucleuscontained
forforebrainonly(Table2).Ofthese10cases,sixwiththe
low to moderate labeling. Moderate to dense labeling was
highest numbers of retrogradely labeled neurons in the
found in the dendritic region of the locus coeruleus (the
SCN are described in detail below. An example of retro-
dendriticregionalsooverlapswithBarrington’snucleus),with
gradely labeled neurons in the SCN following a large in-
manylabeledfibersintermingledwithTH-positivedendrites.
jectionintheMPAisshowninFig.2A(case482).
Onlylowtolow-moderatelabelingwasseeninthecellbody
Anterogradelabelinginthecholinergiccellregions. As regionofthelocuscoeruleus.
shown in Table 2, of the six injections in the MPA that Of the two MPA injections resulting in light retro-
producedstrongretrogradelabelingintheSCN,fourros- grade labeling in the SCN, the rostral injection (504)
tral injections (472, 473, 481 and 482) produced denser showed denser labeling in the dorsal and ventral tube-
labeling of varicose axons in the substantia innominata romammillary nuclei and in the dendritic region of the
(moderate to moderate-dense; Fig. 2B) than in the other locus coeruleus, than in the other regions examined
three basal forebrain regions examined (mostly sparse).
(Table2);thisisconsistentwiththeaboveresults.With
TwocaudalMPAinjections(501and503)yieldedsparse
the caudal injection of the two (453), the dorsal tube-
to low density labeling across all the basal forebrain
romammillary nucleus contained the densest of all the
regions.
anterograde labeling.
Four MPA cases provided data primarily based on
BDAlabeling(406,407,453and504).AsBDAisprimarily Anterogradelabelinginthereticularformation. Sparse
an anterograde tracer, these cases showed little or no ornoanterogradelylabeledfiberswereseeninthemesen-
Table2.ThenumbersofretrogradelylabeledneuronsintheSCNandthedensitiesofanterogradelylabeledvaricosefibresincholinergic,orexin,andaminergiccellgroupsfollowinginjectionsof
BDAorBDA(cid:1)CTBintheMPAa
Case 504 481 472 473 482 406 453 407* 503 501
Tracerinjection
Site1 MPA MPA MPA MPA MPA MPA MPA,PVH BST(MPA, MPA,PVH MPA,PVH
(LS) (LS) (LS) (BST) (Re) AH) (AH,Re) (AH,Re)
AP (cid:5)0.3 (cid:5)0.3 (cid:5)0.3 (cid:5)0.4 (cid:5)0.8 (cid:5)0.92 (cid:5)1.3 (cid:5)1.3 (cid:5)1.4 (cid:5)1.4
ML 0.2 0.4 0.6 0.2 0.4 0.8 0.1 0.9 0.4 0.4
DV 7.8 8.6 8.6 8.6 8.6 7.8 7.6 8.0 8.2 8.6
Size M L M/L M/L L L M S L L
Tracers BDA(cid:1)CTB BDA(cid:1)CTB BDA(cid:1)CTB BDA(cid:1)CTB BDA(cid:1)CTB BDA BDA BDA BDA(cid:1)CTB BDA(cid:1)CTB
Numbersofretrogradelylabeledneurons2 S
SCNshell (0) 16 39 58 25 0 0 (4) (2) .D
SCNcore (1) 10 13 16 2 0 3 0 (6) (4) eu
rv
Densitiesofanterogradelabelinginwake-relatedcellgroups3 eilh
Cholinergiccellgroups e
Medialseptum–verticaldiagonalband n.a. (cid:1) n.a. n.a. (cid:5)/(cid:1) (cid:1)(cid:1)/(cid:1)(cid:1)(cid:1) (cid:5)/(cid:1) (cid:1) (cid:1) (cid:1) ra
n
Horizontaldiagonalband (cid:1) (cid:1)/(cid:1)(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:1) (cid:1) d
Magnocellularpreopticnucleus (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5) (cid:5) (cid:5)/(cid:1) (cid:5)/(cid:1) K.
Substantiainnominata (cid:1) (cid:1)(cid:1)/(cid:1)(cid:1)(cid:1) (cid:1)(cid:1) (cid:1)(cid:1) (cid:1)(cid:1)/(cid:1)(cid:1)(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:1) (cid:5)/(cid:1) Se
Pedunculopontinetegmentalnucleus (cid:5) n.a. (cid:5) (cid:5) n.a. n.a. (cid:5) n.a. (cid:5)/(cid:1) (cid:5)/(cid:1) m
b
Laterodorsaltegmentalnucleus (cid:5)/(cid:1) n.a. (cid:5)/(cid:1) (cid:5)/(cid:1) n.a. n.a. (cid:5)/(cid:1) n.a. (cid:5)/(cid:1) (cid:5)/(cid:1) a
/
N
Orexin/hypocretin-containingcellgroup e
u
Orexinfield,medial (cid:1)(cid:1)(cid:1) (cid:1)(cid:1)(cid:1) (cid:1)(cid:1)/(cid:1)(cid:1)(cid:1) (cid:1)(cid:1)/(cid:1)(cid:1)(cid:1) (cid:1)(cid:1)(cid:1) n.a. (cid:5) n.a. (cid:1)(cid:1)/(cid:1)(cid:1)(cid:1) (cid:1)(cid:1)/(cid:1)(cid:1)(cid:1) ro
Orexinfield,lateral (cid:1)(cid:1) (cid:1)(cid:1) (cid:1)(cid:1) (cid:1) (cid:1)(cid:1) n.a. (cid:5) n.a. (cid:1)/(cid:1)(cid:1) (cid:1) sc
ie
n
c
Aminergiccellgroups e
Tuberomammillarynucleus,dorsal (cid:1)(cid:1) (cid:1)(cid:1) (cid:1)(cid:1) (cid:1)(cid:1) (cid:1)(cid:1) n.a. (cid:1)(cid:1) n.a. (cid:1)(cid:1) (cid:1)(cid:1) 13
Tuberomammillarynucleus,ventral (cid:1)(cid:1) (cid:1) (cid:1)(cid:1) (cid:1)/(cid:1)(cid:1) (cid:1) n.a. (cid:1) n.a. (cid:1) (cid:1)(cid:1) 0
Ventraltegmentalarea (cid:1)/(cid:1)(cid:1) n.a. (cid:5)/(cid:1) (cid:5)/(cid:1) n.a. n.a. (cid:5)/(cid:1) n.a. (cid:1) (cid:1) (20
Substantianigra,parscompacta (cid:5)/(cid:1) n.a. (cid:5)/(cid:1) (cid:5)/(cid:1) n.a. n.a. (cid:5)/(cid:1) n.a. (cid:5)/(cid:1) (cid:1) 05
)
Dorsalraphenucleus (cid:1) n.a. (cid:1)/(cid:1)(cid:1) (cid:1) n.a. n.a. (cid:5)/(cid:1) n.a. (cid:1) (cid:1)(cid:1) 1
6
Locuscoeruleus,dendriticregion (cid:1)(cid:1) n.a. (cid:1)(cid:1)(cid:1) (cid:1)(cid:1)/(cid:1)(cid:1)(cid:1) n.a. n.a. (cid:5)/(cid:1) n.a. n.a. (cid:1)(cid:1) 5
–
Locuscoeruleus,cellbodyregion (cid:1) n.a. (cid:1) (cid:1) n.a. n.a. (cid:5)/(cid:1) n.a. n.a. (cid:1)/(cid:1)(cid:1) 18
3
Othercellgroups
Mesencephalicreticularformation (cid:5)/(cid:1) n.a. (cid:5)/(cid:1) (cid:5)/(cid:1) n.a. n.a. (cid:5) n.a. (cid:5)/(cid:1) (cid:5)/(cid:1)
Pontinereticularformation (cid:5) n.a. (cid:5)/(cid:1) (cid:5) n.a. n.a. (cid:5) n.a. (cid:5)/(cid:1) (cid:5)/(cid:1)
aInjectionsitesandretrogradelabelingdataareadaptedfromDeurveilheretal.(2002).CaseswithstrongretrogradelabelingintheSCNandstronganterogradelabelinginatargetareindicated
inboldnumbersinthefirstrow,andtheprominentpartsoftheirdataareshaded.CTBwasusedataconcentrationof0.25%inallcasesexceptforcases472and473,inwhichCTBwasused
at0.5%.BDAwasusedattheconcentrationof10%inallcases.Theasteriskforcase407indicatesthattheinjectionsitewasa“miss,”i.e.,centeredoutsideoftheMPA.
1Majorand,inparentheses,minorstructuresinvolvedintheinjectionsitesareindicated.Coordinatesindicatethelocationofthecenterofthecoreofeachinjectionsite:Anterior-posterior(AP)from
bregma;mediolateral(ML)frommidline;dorsoventral(DV)fromskullsurface(inmm).Sizeofinjection:L,large;M,medium;S,small.
2ThenumberofretrogradelylabeledneuronsintheSCNisbasedonanaverageofcountsatthreerostrocaudallevels.ForthecasesusinginjectionsofaBDA/CTBmixture,theresultswithCTB
labelingareshown;whentheCTBretrogradelabelingcouldnotbedeterminedfortechnicalreasons,theresultswithBDAlabelingareshowninparentheses.n.a.,notavailable;n.d.,notdetermined.
3ThedensitiesofvaricosefibersanterogradelylabeledwithBDAincholinergic,orexin/hypocretin-containingandaminergiccellgroupswerebasedonanaverageovertwoorthreelevels:(cid:5)no
1
labeling;(cid:1)lowdensity;(cid:1)(cid:1)moderatedensity;(cid:1)(cid:1)(cid:1)densedensity. 69
170 S.DeurveilherandK.Semba/Neuroscience130(2005)165–183
Fig.2. ExamplesofretrogradelabelingintheSCNandanterogradelabelinginselectedcentersofthearousalsystemfollowingBDA(cid:1)CTBinjection
into the MPA (A–D; case 482) and the SPVZ (E–H; case 517). All images are ipsilateral to the injection site (i.e. right side of the brain).
PhotomicrographsoftheinjectionsiterevealedforCTBandforBDAincase482havebeenshowninDeurveilheretal.(2002).(A)IntheSCN,
CTB-labeledneurons(arrows)aredistributedmostlyintheshellratherthanthecoreregion.Theshellandcoreregionsaredepictedindottedlines
basedonvasopressinandNPYimmunoreactivity,respectively,fromaseparateseriesofsections.Severalretrogradelylabeledneuronsarealsoseen
inadjacentregions,particularlydorsomedialtotheSCN.(B)Inthebasalforebrain,BDA-labeledfibersandterminals,ratedas“moderate-dense”in
density,arepresentinthesubstantiainnominata.Mostterminalsappeartobeoftheenpassanttype.(C)Intheperifornicalarea,BDA-labeledvaricose
axons(arrows)aremixedwithpreprohypocretin-irororexinperikarya(browncells).(D)Inthedorsaltuberomammillarynucleus,manyBDA-labeled
bouton-likeswellings(arrows)arecloselyassociatedwithHDC-irorhistaminergicneurons(browncells).TheinsetshowsonesuchHDC-irneuron
(arrowhead)alongwithacloselyapposingBDA-labeledvaricoseaxonatahighermagnification.(E)BDAinjectionsiteintheSPVZ.Theholeatthe
centeroftheinjectionsiteisanartifactduetotissuecrack.(F)IntheSCN,CTB-labeledneuronsarepresentinboththeshellandcoreregions
(examplesoflabeledneuronsindicatedbyblackandwhitearrows,respectively).NotethattheSCNcorecontainsNPY-irfibers(brown).(G)Inthe
perifornicalarea,mostBDA-labeledterminals(black)donotshowobviousassociationwithorexinergicorpreprohypocretin-irperikarya(browncells).
Aretrogradelylabeledneuronisalsopresent(blackcell).(H)Inthelocuscoeruleus,afewBDA-labeledfibers(arrows)areseeninthedendritic(LCd)
andthecoreregionsofthelocuscoeruleus(LCc;brownsomataanddendrites)visualizedwithTHimmunoreactivity.Forotherabbreviations,see
Table1.Scalebars(cid:2)100(cid:3)m(A–D,F);500(cid:3)m(E);50(cid:3)m(G,H);20(cid:3)m(insetinD).
Table3.ThenumbersofretrogradelylabeledneuronsintheSCNandthedensitiesofanterogradelylabeledvaricosefibresincholinergic,orexin,andaminergiccellgroupsfollowinginjectionsof
BDAorBDA(cid:1)CTBintheSPVZa
Case 402 400 517 471 399 470 518 485 499 511 498*
Tracerinjection
Site SPVZ, PVH, SPVZ, SPVZ, SPVZ PVH, SPVZ, PVH, SPVZ,AH, SPVZ,AH, AH
RCh, SPVZ, PVH(AH, PVH(AH, (PVH) SPVZ(AH, VMH,AH, SPVZ(AH, PVH(Re) PVH (SPVZ)
PVH (AH, VMH,Re, Re) Re,AM) PVH Re,AM) (DMH,Re)
(MPA, Re, AM) (DMH,Re)
AH) AM)
AP (cid:5)1.6 (cid:5)1.8 (cid:5)1.8 (cid:5)1.8 (cid:5)2.12 (cid:5)2.12 (cid:5)2.12 (cid:5)2.12 (cid:5)2.12 (cid:5)2.12 2.3
ML 0.4 0.2 0.3 0.6 0.1 0.2 0.4 0.6 0.8 0.8 0.8
DV 9.2 8.0 8.7 8.2 9.4 8.3 9.2 8.2 8.2 8.4 8.6 S
.
Size L M M S/L M M/L L L M L S D
Tracers BDA BDA BDA(cid:1)CTB BDA(cid:1)CTB BDA BDA(cid:1)CTB BDA(cid:1)CTB BDA(cid:1)CTB BDA(cid:1)CTB BDA(cid:1)CTB BDA(cid:1)CTB eu
rv
Numbersofretrogradelylabeledneurons eilh
SCNshell 16 1 13 4 14 (1) (6) (0) (0) 17 (0) e
r
SCNcore 22 2 10 5 9 (3) (4) (0) (0) 12 (0) a
n
d
Densitiesofanterogradelabelinginwake-relatedcellgroups K
.
Cholinergiccellgroups S
Medialseptum-–verticaldiagonalband (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) n.a. (cid:5)/(cid:1) n.a. (cid:1) (cid:1)/(cid:1)(cid:1) (cid:1)(cid:1) (cid:1) (cid:1)/(cid:1)(cid:1) em
Horizontaldiagonalband (cid:5) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5) (cid:5)/(cid:1) (cid:5) (cid:5)/(cid:1) (cid:1)/(cid:1)(cid:1) (cid:1)(cid:1) (cid:5)/(cid:1) (cid:1) ba
Magnocellularpreopticnucleus (cid:5) (cid:5) (cid:5)/(cid:1) (cid:5) (cid:5) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) /
N
Substantiainnominata (cid:5)/(cid:1) (cid:5) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) e
u
Pedunculopontinetegmentalnucleus n.a. n.a. (cid:5) n.a. n.a. n.a. (cid:5)/(cid:1) (cid:1) (cid:5) (cid:5) n.a. ro
s
Laterodorsaltegmentalnucleus n.a. n.a. (cid:5)/(cid:1) n.a. n.a. n.a. (cid:5)/(cid:1) (cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) n.a. c
ie
n
Orexin/hypocretin-containingcellgroup ce
Orexinfield,medial n.a. n.a. (cid:1)(cid:1) (cid:1)/(cid:1)(cid:1) n.a. (cid:1)(cid:1) (cid:1)(cid:1) n.d. (cid:1)(cid:1) (cid:1)(cid:1)(cid:1) n.a. 1
3
Orexinfield,lateral n.a. n.a. (cid:1) (cid:1) n.a. (cid:5)/(cid:1) (cid:1) n.d. (cid:1) (cid:1)(cid:1) n.a. 0
(2
0
Aminergiccellgroups 0
5
Tuberomammillarynucleus,dorsal n.a. n.a. (cid:1)(cid:1)(cid:1) (cid:1)/(cid:1)(cid:1) n.a. (cid:1)/(cid:1)(cid:1) (cid:1)(cid:1) (cid:1)(cid:1) (cid:1) (cid:1)(cid:1) n.a. )
1
Tuberomammillarynucleus,ventral n.a. n.a. (cid:1)(cid:1) (cid:5)/(cid:1) n.a. n.d. (cid:5)/(cid:1) (cid:1)/(cid:1)(cid:1) (cid:1) (cid:1) 65
Ventraltegmentalarea n.a. n.a. (cid:5)/(cid:1) n.a. n.a. n.a. (cid:5)/(cid:1) (cid:1) (cid:5) (cid:5)/(cid:1) n.a. –1
Substantianigra,parscompacta n.a. n.a. (cid:5)/(cid:1) n.a. n.a. n.a. (cid:1) (cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) n.a. 83
Dorsalraphenucleus n.a. n.a. (cid:1) n.a. n.a. n.a. (cid:5)/(cid:1) (cid:1) (cid:1) (cid:5)/(cid:1) n.a.
Locuscoeruleus,dendriticregion n.a. n.a. (cid:1)/(cid:1)(cid:1) n.a. n.a. n.a. (cid:1)/(cid:1)(cid:1) (cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) n.a.
Locuscoeruleus,cellbodyregion n.a. n.a. (cid:1) n.a. n.a. n.a. (cid:5)/(cid:1) (cid:5) (cid:5) (cid:5) n.a.
Othercellgroups
Mesencephalicreticularformation n.a. n.a. (cid:5)/(cid:1) n.a. n.a. n.a. (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) n.a.
Pontinereticularformation n.a. n.a. (cid:5) n.a. n.a. n.a. (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5) (cid:5)/(cid:1) n.a.
aTheinjectionsitesandretrogradelabelingdataareadaptedfromDeurveilheretal.(2002).CaseswithstrongretrogradelabelingintheSCNandstronganterogradelabelinginatargetareindicatedinboldcase
numbersinthefirstrow,andtheprominentpartsoftheirdataareshaded.Theasteriskforcase498indicatesthattheinjectionwasa“miss”andwascenteredoutsideoftheSPVZ.CTBwasusedataconcentration
of0.25%inallcasesexceptforcases470and471,inwhichCTBwasusedat0.5%.EachnumberofretrogradelylabeledneuronsintheSCNrepresentsanaverageofcountsfromthreelevelsexceptforcases
400,499and518,inwhichanaverageofcountsfromtwolevelswasused.SeefootnotestoTable2forfurtherdetails.
1
7
1
172 S.DeurveilherandK.Semba/Neuroscience130(2005)165–183
Fig.3. PhotomicrographsshowingtheanterogradelabelinginselectedcentersofthearousalsystemfollowingBDA(cid:1)CTBinjectionintothecaudal
DMH(case520).(A)TherelativelysmallinjectionsiterevealedforBDAislocatedintheventrolateralaspectofthecaudalDMH.(B)Inthelateral
perifornicalarea,BDA-labeledfibers(black)arepresentamongpreprohypocretin-ir(orexin)neurons(brown).(C)Inthedorsaltuberomammillary
nucleus,someBDAbouton-likeswellings(arrows)arecloselyassociatedwithHDC-ir(histaminergic)cellbodiesanddendrites(brown).(D)Inthe
laterodorsaltegmentalnucleus,closeassociationsareseenbetweenBDA-labeledterminals(arrows)andVAChT-ir(cholinergic)cellbodies(brown
cells).(E)Thedendriticregionofthelocuscoeruleus,whichalsooverlapswithBarrington’snucleus,containsmoderatelyabundantBDA-labeled
varicosefibers.(F)ThissectionatthesamelevelasinEwasstainedforTHtovisualizenoradrenergiccellbodiesanddendrites.Forabbreviations,
seeTable1.Scalebars(cid:2)500(cid:3)m(A),100(cid:3)m(B,E,F),50(cid:3)m(C,D).
cephalicandpontinereticularfieldsinanyoftheMPAcases TheSPVZ
(Table2).
TheretrogradelabelingwitheitherCTBorBDAindicated
Insummary,theresultsbasedonbothCTBandBDA
thattheSCNprojectionstotheSPVZregionaroseequally
labeling indicate that the MPA receives strong projec-
from the shell and the core (Table 3). Numerous retro-
tionsfromtheSCNand,inturn,providesstrongprojec-
gradely labeled neurons were also present in the ventral
tions to the orexin field, the tuberomammillary nucleus,
and the locus coeruleus dendritic region (which also part of the lateral septum, bed nucleus of the stria termi-
overlaps with Barrington’s nucleus). The projections to nalis,anteroventralperiventricularandventromedialhypo-
thesubstantiainnominataoriginatemostlyfromtheros- thalamic nuclei (Deurveilher et al., 2002). Anterograde
tralMPA.TheseresultsindicatethattheMPAisastrong BDAlabelingwasanalyzedinfivecasesforbothforebrain
candidate for a relay in respective indirect pathways and brainstem, and in six cases for forebrain only (Table
from the SCN. 3). Of these 11 cases, seven with the largest numbers of
S.DeurveilherandK.Semba/Neuroscience130(2005)165–183 173
retrogradelylabeledneuronsintheSCNaredescribedin GiventhatthemedialpartoftheboxusedtoassessBDA
detail.Theseseveninjectionswerecenteredmedially(ML labelingintheorexinfieldpartlyoverlappedwiththelateral
0.1–0.4) or laterally (ML 0.6–0.8) within the SPVZ. An region of the DMH, the projections of the SPVZ to the
example of a medium-sized injection in the SPVZ, and medial orexin field may be considered to be part of the
retrograde labeling in the SCN after this injection are SPVZ projections to the DMH reported by Watts et al.
showninFig.2EandF,respectively(case517). (1987)andChouetal.(2003).
Anterogradelabelinginthecholinergiccellregions. All Anterogradelabelingintheaminergiccellregions. The
seven SPVZ injections that yielded strong retrograde la- SPVZinjectionsthatresultedinstrongretrogradelabeling
beling in the SCN produced only sparse to low, or no in the SCN produced moderate to dense anterograde la-
anterograde labeling in any of the four basal forebrain belinginthedorsaltuberomammillarynucleus,whichwas
cholinergiccellregionsexamined(Table3).Fiveofthese the densest of all aminergic cell groups examined. Many
seven injections were located medially in the SPVZ (399, anterogradely labeled terminals were found in the vicinity
400, 402, 470 and 517), one was centered in the lateral of HDC-ir cell bodies and dendrites (511, 517 and 518;
SPVZ (511), and the other involved both the medial and Table3).Onlysparsetomoderatelabelingwasseeninthe
lateral SPVZ (518). Three injections that resulted in only ventral tuberomammillary nucleus, and sparse to low an-
minor retrogradely labeled neurons in the SCN (471, 485 terogradelabelingintheventraltegmentalarea,substantia
and 499) were located in the lateral SPVZ, and yielded nigraparscompacta,anddorsalraphenucleus.Theden-
denser labeling in the MS-VDB and HDB (mostly low- driticregionofthelocuscoeruleusshowedsparsetolow-
moderatetomoderate)thaninmorecaudalbasalforebrain moderatelabeling,whilethecoreregionshowedonlylow
regions, with the exception of case 471 which provided or no labeling (Fig. 2H). Similarly, even the SPVZ injec-
sparseornolabelinginanybasalforebrainregionexam- tionsresultinginlittleornoretrogradelabelingintheSCN
ined. A small injection that missed the SPVZ and was produced generally denser anterograde labeling in the
tuberomammillarynucleusthanintheotherbrainstemnu-
centered laterally in the anterior hypothalamic area but
cleiexamined(485and499).
with minor diffusion into the SPVZ resulted in stronger
anterograde labeling in the rostral than the caudal basal Anterograde labeling in the reticular formation. All
forebrain regions (498). In the brainstem, all SPVZ injec- SPVZinjectionsproducedsparseornoanterogradelabel-
tions produced sparse or no anterograde labeling in the ing in the mesencephalic and pontine reticular formation
pedunculopontineandlaterodorsaltegmentalnuclei. (Table3).
Functional differences have been reported between In summary, the SPVZ receives strong projections
theventralandthedorsalSPVZintheirrolesincircadian fromtheSCNand,inturn,sendsstrongprojectionstoboth
rhythms of sleep, locomotion and temperature (Lu et al., the orexin field and tuberomammillary nucleus, indicating
2001),andthereforeweexaminedtheinjectionsitesusing thattheSPVZisastrongcandidaterelaynucleusinthese
thisschemeinanattempttoidentifypossibledifferencesin indirect pathways from the SCN. The SPVZ does not ap-
thepatternofanterogradelabeling.Oftheseveninjections peartobepositionedtorelaytheSCNprojectionsfurther
in the SPVZ that yielded strong retrograde labeling in the caudallytothebrainstem.
SCN, only one injection (case 402) was located in the
ventralSPVZ,whereastheothersixinjectionswereinthe TheDMH
dorsal SPVZ. The density of anterograde labeling within
The retrograde labeling data indicated that the SCN pro-
the basal forebrain cholinergic cell groups in the ventral
jectionstotherostralDMHaroseequallyfromtheshelland
SPVZcase(402)didnotdifferfromthatinthedorsalSPVZ
core, whereas those to the caudal DMH originated pre-
cases.Unfortunately,theanterogradelabelingintheother
dominantly from the shell (Table 4). In addition to SCN
forebrainregionsandthebrainstemincase402couldnot
neurons, numerous retrogradely labeled neurons were
be analyzed for a technical reason, which precluded fur-
foundinthelateralseptum,bednucleusofthestriatermi-
thercomparisons.
nalis, anteroventral periventricular nuclei, anterior hypo-
thalamic nucleus, and dorsal tuberomammillary nucleus
Anterograde labeling in the orexin cell region. The
(Deurveilher et al., 2002). Anterograde BDA labeling was
injectionsintheSPVZresultinginstrongretrogradelabeling
analyzed in five cases for both forebrain and brainstem,
in the SCN produced denser anterograde labeling in the
and in three cases for forebrain only (Table 4). Of these
medial (moderate to dense) than in the lateral (sparse to
eightcases,fivewiththehighestnumbersofretrogradely
moderate)regionoftheorexinfield(470,511,517and518;
labeledneuronsintheSCNaredescribedbelowindetail,
Table3).VaricosefiberslabeledfromtheSPVZwereinter-
whereastheotherthreeproducinglessretrogradelabeling
spersedwithorexin-ircellbodiesanddendrites,particularly
provided supplementary data. An example of a small in-
with the more lateral SPVZ injections (517, Fig. 2G). Simi-
jectionsiteintheDMHisshowninFig.3A(case520).
larly,injectionswithlittleornoretrogradelabelingintheSCN
showeddenserlabelinginthemedialthaninthelateralpart Anterograde labeling in the cholinergic cell regions.
oftheorexinfield(471and499). A small injection that was largely confined to the caudal
ItshouldbenotedthatinjectionsintheSPVZresulted DMH (520) and yielded strong retrograde labeling in the
in moderate to dense anterograde labeling in the DMH. SCNresultedinsparsetolowanterogradelabelinginthe
1
7
4
Table4.ThenumbersofretrogradelylabeledneuronsintheSCNandthedensitiesofanterogradelylabeledvaricosefibersincholinergic,orexin,andaminergiccellgroupsfollowinginjectionsof
BDAorBDA(cid:1)CTBintheDMHa
Case 415 483 490 512 409 520 515 514*
Tracerinjection
Site DMH,DA DMH,DA DMH,VMH DMH DMH DMH DMH PeF
(AH,VMH, (VMH,AH, (DA,AH) (DA,AH, (DA,PH, (PH,TMNd) (DMH)
Re,CM) Re) VMH,Re) Re)
AP (cid:5)2.56 (cid:5)2.8 (cid:5)3.14 (cid:5)3.14 (cid:5)3.3 (cid:5)3.3 (cid:5)3.3 (cid:5)3.3
ML 0.6 0.6 0.6 0.8 0.4 0.8 0.8 1.1
DV 8.6 8.6 9.0 8.8 8.8 9.2 9.2 8.8
S
Size L L L L M S L S .
D
Tracers BDA BDA(cid:1)CTB BDA(cid:1)CTB BDA(cid:1)CTB BDA BDA(cid:1)CTB BDA(cid:1)CTB BDA(cid:1)CTB e
u
rv
Numbersofretrogradelylabeledneurons e
SCNshell 0 2 12 8 4 5 27 1 ilh
e
SCNcore 0 2 13 11 3 2 13 0 r
a
n
d
Densitiesofanterogradelabelinginwake-relatedgroups
K
Cholinergiccellgroups .
S
Medialseptum-–verticaldiagonalband (cid:5)/(cid:1) n.a. (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:1)(cid:1)/(cid:1)(cid:1)(cid:1) (cid:1) (cid:1)(cid:1)(cid:1) (cid:1) e
m
Horizontaldiagonalband (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:1)/(cid:1)(cid:1) (cid:5)/(cid:1) (cid:1)(cid:1)(cid:1) (cid:1) b
a
Magnocellularpreopticnucleus (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:1)/(cid:1)(cid:1) (cid:5)/(cid:1) /
Substantiainnominata (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:5)/(cid:1) (cid:1)(cid:1)(cid:1) (cid:1)(cid:1) Ne
Pedunculopontinetegmentalnucleus n.a. n.a. (cid:5) (cid:5)/(cid:1) n.a. (cid:5)/(cid:1) (cid:1)/(cid:1)(cid:1) (cid:5)/(cid:1) uro
Laterodorsaltegmentalnucleus n.a. n.a. (cid:5)/(cid:1) (cid:1)/(cid:1)(cid:1) n.a. (cid:1)/(cid:1)(cid:1) (cid:1)(cid:1) (cid:1)/(cid:1)(cid:1) sc
ie
Orexin/hypocretin-containingcellgroup nc
e
Orexinfield,medial n.a. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
1
Orexinfield,lateral n.a. (cid:1) n.d. (cid:1)(cid:1)/(cid:1)(cid:1)(cid:1) n.d. (cid:1)(cid:1)/(cid:1)(cid:1)(cid:1) (cid:1)(cid:1)(cid:1) n.d. 30
(2
Aminergiccellgroups 00
Tuberomammillarynucleus,dorsal n.a. (cid:1)(cid:1) (cid:1)(cid:1)/(cid:1)(cid:1)(cid:1) (cid:1)(cid:1)/(cid:1)(cid:1)(cid:1) (cid:1)(cid:1)(cid:1) (cid:1)(cid:1)(cid:1) n.d. (cid:1)/(cid:1)(cid:1) 5)
Tuberomammillarynucleus,ventral n.a. (cid:5)/(cid:1) (cid:1)/(cid:1)(cid:1) (cid:5)/(cid:1) n.d. (cid:1)(cid:1) n.d. (cid:1)/(cid:1)(cid:1) 16
Ventraltegmentalarea n.a. n.a. (cid:1) (cid:5)/(cid:1) n.a. (cid:1)(cid:1) (cid:1)(cid:1)(cid:1) (cid:1)/(cid:1)(cid:1) 5–
1
Substantianigra,parscompacta n.a. n.a. (cid:1) (cid:5)/(cid:1) n.a. (cid:1) (cid:1)(cid:1) (cid:1) 8
3
Dorsalraphenucleus n.a. n.a. (cid:1)/(cid:1)(cid:1) (cid:1)(cid:1) n.a. (cid:1)(cid:1) (cid:1)(cid:1)(cid:1) (cid:1)(cid:1)
Locuscoeruleus,dendriticregion n.a. n.a. (cid:5)/(cid:1) (cid:5)/(cid:1) n.a. (cid:1)(cid:1)/(cid:1)(cid:1)(cid:1) (cid:1)(cid:1)(cid:1) (cid:1)(cid:1)(cid:1)
Locuscoeruleus,cellbodyregion n.a. n.a. (cid:5) (cid:5)/(cid:1) n.a. (cid:1) (cid:1)(cid:1) (cid:5)/(cid:1)
Othercellgroups
Mesencephalicreticularformation n.a. n.a. (cid:1)/(cid:1)(cid:1) (cid:1) n.a. (cid:5)/(cid:1) (cid:1)(cid:1) (cid:1)/(cid:1)(cid:1)
Pontinereticularformation n.a. n.a. (cid:5) (cid:5)/(cid:1) n.a. (cid:5)/(cid:1) (cid:1)(cid:1) (cid:1)
aTheinjectionsitesandretrogradelabelingdataareadaptedfromDeurveilheretal.(2002).CaseswithstrongretrogradelabelingintheSCNandstronganterogradelabelinginatargetareindicated
inboldcasenumbersinthefirstrow,andtheprominentpartsoftheirdataareshaded.Theasteriskforcase514indicatesthattheinjectionwasa“miss”andcenteredoutsideoftheDMH.See
footnotestoTable2forfurtherdetails.