Thalamic Input to Distal Apical Dendrites in Neocortical Layer 1 Is Massive and Highly Convergent

16 Pages·2009·1.8 MB·English

CerebralCortexOctober2009;19:2380--2395
doi:10.1093/cercor/bhn259
AdvanceAccesspublicationFebruary2,2009
Thalamic Input to Distal Apical Dendrites Pablo Rubio-Garrido1,2, FlorPe´rez-de-Manzo1,Ce´sar Porrero1,
Maria J.Galazo1and FranciscoClasca´1
in Neocortical Layer 1 Is Massive and
Highly Convergent 1Department of Anatomyand Neuroscience, School of
Medicine, Auto´noma University, Madrid E28029,Spain and
2DepartmentofBasicMedicalScience,SchoolofMedicine,San
Pablo-CEU University, Madrid E28660, Spain
Inputtoapicaldendritictuftsisnowdeemedcrucialforassociative throughoutthethalamus(‘‘matrix’’orM-typecells).Importantly, D
o
learning, attention, and similar ‘‘feedback’’ interactions in the it was noted that the function of these thalamocortical cells w
n
cerebralcortex.Excitatoryinputtoapicaltuftsinneocorticallayer would probably be different from the better-known cells that lo
a
1 has been traditionally assumed to be predominantly cortical, as primarilytargetcorticallayer4(‘‘core’’orC-type): notonlydid de
d
thalamic pathways directed to this layer were regarded relatively the laminar segregation of the axons imply that each cell type fro
scantanddiffuse.However,thesensitivetracingmethodsusedin would reach different elements of the cortical circuitry but m
tnhuempbreersse(nmtesatund~y4s5h0o0w/mmth2a)t,otfhtrhoaulgahmoouctotrhtiecarlatnenueroocnosrtceoxn,vlearrggee tahsceerendiwnagsinaplsuots,ehviaddenacedifftehraetnteascohmatyspizee,raencde/ivoerdexdpifrfeesrseendt https
inlayer1andthatthisconvergencegivesrisetoaveryhighlocal differentcalcium-bindingproteins(Jones2001). ://a
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densityofthalamicterminals.Moreover,weshowthatthelayer1-- TheM-type/C-typedichotomyhasrecentlybeensingledout ad
e
projectingneuronsarepresentinlargenumbersinmost,butnotall, as a key organizational principle in thalamocortical pathways m
motor, association, limbic, and sensory nuclei of the rodent (Jones 2007). However, information regarding M-type path- ic.o
thalamus.Somelayer1--projectingaxonsbranchtoinnervatelarge ways remains quite scant and fragmentary. There is consider- up
swathsofthecerebralhemisphere,whereasothersarborizewithin able confusion regarding where exactly M-type neurons are .co
m
only a single cortical area. Present data imply that realistic locatedinthethalamus(Llina´setal.2002;Kubotaetal.2007) /c
modeling of cortical circuitry should factor in a dense axonal and how prevalent they are. Likewise, evidence regarding the erc
canopy carrying highly convergent thalamocortical input to strength of M-type input to the cortex is conflicting. On one or/a
pyramidal cell apical tufts. In addition, they are consistent with hand, thalamocortical pathways targeting layer 1 seem to be rtic
thenotionthatlayer1-projectingaxonsmaybearobustanatomical relatively few, as indicated by the small numbers of thalamic le
substrate for extensive ‘‘feedback’’ interactions between cortical neurons labeled by retrograde tracer injections in superficial -ab
s
areasvia thethalamus. cortical layers in comparison to the number labeled by tra
injections involving the middle layers (reviewed in Avendan˜o ct/1
Keywords: cerebral cortex, corticalmicrocircuit, dendrites, pyramidal et al. 1990) and bythe laminar patternof the cortical labeling 9
neuron, thalamus,top--downinteractions produced by a tracer deposit in most thalamic nuclei /10
/2
(Herkenham 1986; Jones 2007). Moreover, individual thalamic 3
8
axons in layer 1 reportedly have relatively few branches and 0/6
Introduction sparseboutons(FersterandLeVay1978;Arbuthnottetal.1990; 001
Lu and Lin 1993; Portera-Cailliau et al. 2005). On the other 7
3
A prominent feature of neocortical organization is that the hand, recent immunolabeling observations for the thalamo- b
y
apicaldendritictuftsofpyramidalneuronsaccumulateinlayer corticalterminalmarkervesicularglutamatetransportertype2 g
u
1.Inputsreachingthesetuftsareincreasinglyviewedascrucial (VGluT2; Fujiyama et al. 2001; Kubota et al. 2007) seem to e
s
for the ‘‘feedback’’ interactions in the cerebral cortex that imply that thalamic innervation of layer1is substantial. t o
n
underlie cognitive processes such as associative learning and Here,weexaminedtheoriginandconvergence/divergence 0
6
attention (Sjo¨stro¨m and Ha¨usser 2006; Gilbert and Sigman of M-type pathways in the rat cerebral cortex using sensitive A
2co0n07si)d.erEexdcittoatboerypriendpoumtintoantnlyeoccoortritcicaall(Claayuellrer119i9s5g;Denoeurgallalys pceatllhswcaoyn-tsrtaictuinteg aandvearnyalsyisziasbmleetphoopdusl.aWtioenfooufndthethatthaMla-mtyupse. pril 20
and Martin 2007). Furthermore, we demonstrate that M-type cell innervation in 19
In addition, it has been known for some time that some cortical layer 1 is both massive and highly convergent, in
thalamic axons arborize in layer 1 (Lorente de No 1938; combinations that are specific for the various areas. We also
Killackey and Ebner 1973; Herkenham 1979), but these were show that whereas many M-type axons branch over wide
widely assumed to represent a relatively minor and diffuse swaths of the cerebral hemisphere others arborize within
(‘‘nonspecific’’; Lorente de No 1938) input to cortex. These a singlearea.
layer1--directedpathwayshavebeenpersistentlyassociatedin
theliteraturewiththeintralaminarandmidlinethalamicnuclei,
despite substantial evidence to the contrary (for reviews, see
Materials and Methods
Herkenham 1986;Avendan˜oet al. 1990).
Some years ago, fragmentary observations in carnivores and
AnimalsandAnestheticProcedures
primates led to the hypothesis (Avendan˜o et al. 1990; Jones
The brains from a total of 75 adult (4--12 months of age) female
1998) that the thalamocortical layer 1--projecting neurons Sprague--Dawley rats obtained from the School of Medicine Animal
mightactuallyconstituteasubstantialcellpopulationoriginating Facilitywereusedforthevariousexperimentsreportedinthisstudy.
(cid:1)TheAuthor2009.PublishedbyOxfordUniversityPress.Allrightsreserved.
Forpermissions,pleasee-mail:[email protected]
From these, the 37 animals with one or more tracer deposit experi- cryoprotected, and coronally sectioned as described above. For
ments valid for analysis are listed in Supplementary Table SM1. reference, the cerebral hemispheres from 2 additional unoperated
Procedures were carried out in accordance with the Society for animalswereflattenedinthesameway.
Neuroscience Handbook on the Use of Animals in Neuroscience ThalamicontainingtheBDAinjectionsiteswerecoronallysectioned
ResearchandtheEuropeanCommunityCouncilDirective86609and andprocessedwithastandardavidin--biotin--peroxidasecomplex(ABC;
were approved by our university’s Bioethics Committee. All surgical Vector Laboratories, Burlingame, CA) + diaminobenzidine (DAB)
procedureswereconductedunderacombinationofketamine(66mg/kg, protocol. Sections were finally mounted on gelatin-coated slides, air-
intramuscularly [i.m.]) and xylazine (8 mg/kg, i.m.). Additional partial dried, thionin counterstained, and coverslipped. Instead of counter-
doses were administered as required during the surgical procedure to staining,in2cases,thalamicsectionswerehistochemicallyprocessed
keeptheanimalareflexic,butbreathingspontaneously.Throughoutthe torevealCyOactivity.
postoperative period,animalsweregivenamoxicillin (3mg/kg/day).At TorevealtheanterogradelytransportedBDAinsubpialaxonarbors,
thetimeofsacrifice,animalswereoverdosedwithsodiumpentobarbital frozensectionsof theflattenedhemisphereswere cut parallel to the
(80mg/kg,intraperitoneally). pia mater at 60 lm. Labeling was developed on the free-floating
sections with ABC + DAB. Sections were then mounted, thionin
counterstained, and coverslipped with DePeX. The cerebral hemi- D
o
DeliveryofAxonalTracers spheres from the 2 brains that had been flattened and fixed without w
To retrogradely label the somata of all thalamocortical neurons tracer injections were tangentially sectioned at 60 lm, processed to nlo
reveal CyO activity, and mounted and coverslipped as above. This a
innervating a given zone in layer 1, we made epipial applications of d
Fast Blue (FB, diamidino compound 253/50, Dr Illing GmbH and Co. labelingallowedthedelineationofcorticaldomainssuchasthebarrel ed
KG, Groß-Umstadt, Germany) or Diamidino Yellow Dihydrochloride fieldintheprimarysomaticcortexortheprimaryauditoryandvisual fro
areas,whichcouldthenbecorrelatedwiththepositionofthemedial m
(DY, Dr Illing). Surgical procedures have been described in detail
cerebralarteryanditsbranchesinthetangentialsections. h
elsewhere(Rubio-Garridoetal.2007)andwillonlybebrieflyexplained Intheremaining4animalsthathadreceivedBDAinjectionsinthe ttp
here. Anesthetized animals were positioned in a stereotaxic frame. A s
unilateralcraniotomywasmade,andoneormoresmalltracer-soaked thalamus, labeled axons were directly developed ‘‘in toto,’’ without ://a
tissuesectioning.Thesewholeflattenedhemisphereswereincubated c
piecesoffilterpaperwereplacedonthepiamateroftheneocortex. a
Thebonedefectwasthenoccluded,theskinandmusclesutured,and freefloatingin2%Triton+ABCinPBfor4h,washed,andsubsequently de
stainedusingastandardDABprotocol. m
theanimalsallowedtorecover. ic
To anterogradely label thalamocortical axons in layer 1, we made .o
u
unilateral iontophoretic microinjections of 10 000 Da lysine-fixable p
RetrogradeFluorescentLabelingAnalysis .c
biotinylated dextran-amine (BDA, Molecular Probes-Invitrogen, o
Fluorescent labeling was examined with a Nikon 600 Eclipse m
Carlsbad, CA) in the thalamus. Animals were operated as above. A microscope under 4--403 Wide-Diameter Plan-Apochomat Nikon /c
borosilicate micropipette (10- to 25-lm--inner diameter tip; FHC, e
objectivesanda100-WHgUVlightsource.UsingaNikonDMX1200 rc
Bowdoin,ME)loadedwitha10%solutionofBDAin0.01Mphosphate o
buffer(PB),pH7.4,wasstereotaxicallypositionedintotheventrome- digital camera, high-resolution (1490 dpi) RGB TIFF epifluorescence r/a
dniuacl,lethi.eUlastinegralapoMsitdegraormdeCdiuarl,reonrtthSeoudrocresal(Slatoteerlatilngge,nWicuoloadteDthaalela,mILic) i1(mP0r3aogsoecbsajnfeo,crtPievraieocarhnSdsceaiceUtnioVtinfi2cAinNIneiksvteorrnuymfivletaenlirtdss,ecCta.saBemywbmreierdaegneasc,oqUfuKairm)eddorttoihvreriozneudbgyhstatthhgeee rticle-a
squarepulsesofa2lAcurrentweredeliveredfor10min(7son/off b
Analysis software (Soft Imaging Systems, Mu¨nster, Germany) that s
cycles). The pipette was left in place for another 15 min and then controls both image acquisition and microscope stage motion, 24--32 tra
removed.Boneandskinwereclosedasabove. epifluorescence high-resolution images were automatically assembled ct/1
into a single panoramic view of the thalamus (~100 Mb JPG file). By 9
/1
zooming into these brightly fluorescent images, a thorough high- 0
PerfusionandHistology magnificationanalysiscouldbecarriedoutwithoutanyphotobleaching. /23
Intheexperiments withretrogradefluorescenttracers,animalswere Because the excitation and emission curves of FB and DY overlap 80
allowedtosurvivefor7daysaftertheFBapplication;theywerethen extensively,wefounditimpossibletoseparatebothtracersconsistently /6
0
sacrificed andperfusedthroughtheleftventriclewithsaline(5min) andunambiguouslyinourmaterialusingaLeicaSP2spectralconfocal 01
followed by cold (~4 (cid:2)C) 4% paraformaldehyde in PB for 45 min. microscope. Whereas heavily double-labeled cells could be readily 73
Subsequently,brainswereblockedalongacoronalplane,removedfrom identified by the preferential localization of DY or FB to either the b
y
theskull,andpostfixedbyimmersioninthesamefixativefor24hat nucleusorthecytoplasm,doublelabelinginweaklylabeledcellswas g
u
4(cid:2)C.Tissueblockswerecryoprotectedbysoakinginasucrosesolution oftenuncertain.Asweaklylabeledcellsrepresentedasignificantfraction e
s
(30% in PB) until they sank. Parallel series of 40-lm-thick coronal ofthecellslabeledinourexperiments,wechosetokeepthedouble- t o
sectionswereobtainedonaLeicafreezingmicrotome.Oneseries(1in labeling analysis qualitative to avoid a substantial underestimation n 0
every 5 sections, spaced 160 lm) was immediately mounted onto (Schofieldetal.2007).Therefore,presentdatareflectonlythepresence 6
gelatin-coatedglassslides,air-dried,dehydrated,andcoverslippedwith or absenceof unambiguously double-labeled cells in a giventhalamic Ap
DePeX(Serva,Heidelberg,Germany).Toallowforaprecisedelineation nucleusbutbynomeanstheactualnumberofdouble-labeledcells. ril 2
of thalamic nuclei and cortical areas, additional parallel series were Acompleteseriesofsectionscontainingthecorteximpregnatedby 0
1
stained with cresyl violet or else histochemically reacted for either the epipial deposit was examined. Deposits produced an intense but 9
acetylcholinesterase (AChE; Geneser-Jensen and Blackstadt 1971) or superficially restricted tracer impregnation. Any deposit experi-
cytochrome oxidase (CyO; Wong-Riley et al. 1979) activities. Finally, mentsshowingsignsoftracerspreadintocorticallayer2werediscarded
sectionsweremounted,dehydrated,andcoverslippedasabove. (n = 11). Thus, a total of 32 valid different FB or DY epipial deposit
Thebrainof3animalsinjectedinthethalamuswithBDAwerefixed experimentswereanalyzedforthalamiclabeling(seelistinSupplemen-
andcoronallysectionedasdescribedforFBexperiments(Supplemen- tary Table SM1). In addition, the depth of tracer uptake was further
taryTableSM1).Intheremaining9BDA-injectedanimals,thecerebral assessedfromthepatternofretrogradelabelinginlayers2--7:massivecell
hemisphereswereflattenedtoallowthevisualizationoflabeledaxon somatalabelinginlayers2--3,5,and7andavirtualabsenceoflabelingin
arbors on a plane that was parallel to the pial surface. To this end, layers 4 and 6. This pattern reflects the presence in layer 1 of apical
following the initial saline perfusion, the animals were perfused with dendrites/axonterminalsthathaveoriginatedinthelabeledlayers(Fig.
1%paraformaldehydeinPBfor3minfollowedby3 minofPBalone. 1C;Caulleretal.1998;Rubio-Garridoetal.2007).
Afterremovalfromtheskull,thecerebralhemisphereswereseparated Insomeexperiments,extensivedepositscoveringawideareaofthe
from the diencephalon, and the hippocampus and striatum were dorsalandlateralcorticesweremadeandrevealedalargefractionof
removed.Thewholecerebralcortexwasthenplacedbetween2glass the M-type cell population in a single experiment (Fig. 1E--F). Other
slidesandgentlypressedtoflattenit,afterwhichitwasimmersedin experiments involved small single or double FB/DY deposits in
cold4%paraformaldehydefor48h.Thediencephalonwaspostfixed, different points of the cerebral hemisphere. The labeling patterns
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Figure1. Epipialfluorochromeapplicationsselectivelyimpregnateapatchoflayer1.(A--C)Arepresentativeexampleofasmalltracerdepositconfinedtocerebralcortexlayer1. 0
/2
AdjacentcoronalsectionstakenatthecenterofasmallDYdeposit(Case18b)intheprimaryvisualcortex.Panel(A)showsaNissl-stainedsection.Panel(B)showsthe 3
8
epifluorescenceimageofanadjacentsection.Noticetheheavilyimpregnatedneuropilinthesuperficialpartoflayer1.Atsomedistancebelowthiszone,numerouscellsomata 0
arelabeledinlayers2--3,5,and7.Incontrast,fewornocellsarelabeledinlayers4and6.Inpanel(C),alinediagrambasedonpanel(B)showshowanepipialdepositworks /60
andwhythelaminarpatternofbacklabelingobservedindeeperlayersindicatesthattraceruptakeoccurredonlyinlayer1.Tracerdiffusingfromapieceoftracer-soakedfilter 01
paper(wavythicklineatthetop)appliedonthepiamater-impregnatedlayer1(grayshade).Pyramidalneuronswhoseapicaldendrites(layers2,3,and5)oraxons(layer7) 73
arborizeselectivelyinlayer1areheavilylabeled.Incontrast,layer4or6cells,knowntonotextendaxonsordendritessignificantlyintolayer1,remainunlabeled.(D--E)Adjacent b
y
coronalsectionstakenatthecenterofanFBdeposit(Case20)involvingaregionoftheCg1cingulateandM2motorareas.Thelaminardistributionofcellslabeledunderneath g
the impregnated part of layer 1 is equivalent to that in panels (A--C); the narrow unlabeled band at the layer 3--5 border represents layer 4 in these agranular areas. (F) ue
EpifluorescenceimageofacoronalsectionfromacerebralhemispherethatreceivedanextensiveepipialdepositofFB(Case41).Thepatternofcellsomatalabelingunderthe st o
depositagainrevealsthattraceruptakewaslimitedtolayer1.(G)Representationona‘‘flat’’corticalsurfacemapoftheextentofthe3depositsshowninpanels(A--F).For n
reference,adiagramofaconventionallateralviewoftheratcerebralhemisphereisshown.Intheflatcortexmap,thepialsurfaceofthecerebralhemispherewas‘‘unfolded’’by 0
6
seriallyaligningtheperimetermeasurementsfromallthecorticalcoronalsectionsofPaxinosandWatsonratbrainatlas(PaxinosandWatson2005).Theperimeterswereplotted A
omnetaosuvreermticeanltspawraelrleelslpinaecsedalaigcnceodrdianlgontgotthheeireddisgteandcievidfrionmg tthheeinmteedraiaulraalncdorloantealropdlaonrsea.lThaespoelfcatcstoorfytpheeducnecrelebirsalcuhtem(aisstpehriesrke).(Gthraicykshdaadsheeddarleinaes).reAplroensgentthtisheaexxist,enpteorifmlaeyteerr pril 2
0
1impregnationineachofthe3experimentsillustratedinpanels(A--F).SeeTable1forabbreviations.Bar5500lm. 1
9
produced by these small injections were used to estimate the not explored, as we found them to be practically inaccessible using
convergenceofM-typeaxonsaswellastoextendthemappingofM- thistechnique.
typeneuronstocorticalregionsthatwerenotexploredbythesingle Foreachvalidcase,thalamiclabelingwasexaminedanddrawnfrom
largedeposits(Fig.1A--E,G,seealsoSupplementaryFig.SM1). acompleteseriesofsections.Usingvectorialgraphicssoftware(Canvas
Forcomparisonbetweenexperiments,thezoneoflayer1thatwas X, ACD Systems, Saanichton, British Columbia, Canada) on Apple
impregnatedineachofthevalidexperimentswasreconstructedfrom computers,nuclearbordersweredrawnbysuccessivelyoverlayingthe
the serial coronal sections onto a standard ‘‘flat’’ map of the pial digitaldrawingfromthesectioncontainingthelabelingontothalamus
surface of the whole cerebral hemisphere (Fig. 1F; Paxinos and imagesfromadjacentNissl-,AChE-,andCyO-stainedsections.Because
Watson2005).Ourvaliddepositssampledmostofthemotor,somatic, manyofthelabeledcellsweresituatedatornearnuclearboundaries,
auditory,andvisualcortices,aswellasportionsofthemedialfrontal, thisprocedurewascrucialforconsistentlyascribingthelabeledcellsto
retrosplenial,andinsularcortices;alltold,theycoverednearly60%of agivennucleus.WefollowedtheatlasofPaxinosandWatson(2005)
the neocortical layer 1 surface. Only the frontal and occipital poles for nomenclature and delineation of nuclei. The position of every
andsomezonesofthemedialcortexoraroundtherhinalsulcuswere labeled cell soma was plotted onto the digital drawings from the
2382 ThalamocorticalInputtoApicalDendriticTufts d Rubio-Garridoetal.
images. Drawn cells were automatically counted using the software anteromedial, posterior, and lateral posterior thalamic nuclei,
tools. andmanycellswerealsolabeledinthedorsallateralgeniculate,
laterodorsal, reuniens, medial geniculate, and anterior intra-
BDALabelingAnalysis laminar nuclei. In spite of the large numbers of cells labeled,
ThelocationandextentoftheBDAinjectionsitesinthethalamuswas theirdistributionwasfarfromuniform.Forexample,innuclei
reconstructedfromtheserialcoronalsectionsofthediencephalon. such as the ventrolateral or dorsal lateral geniculate nuclei,
In the flat-sectioned cerebral hemispheres, BDA-labeled thalamo-
labeled cells were densely concentrated in one portion of the
cortical axons were examined and photographed using bright- and
nucleus, whereas other parts were almost unlabeled. Other
dark-fieldoptics.Axonsinlayer1werereconstructedfromserialdigital
nucleithatwerevirtuallyunlabeledweretheventralposterior,
images taken with the motorized microscope stage as described. We
useddark-fieldopticstobettervisualizethesethinaxons.Imageswere dorsomedial, anterior, and gelatinous nuclei. Absence of
takenwiththe103objectivetomaximizethedepthoffocus.Tocover labeling in the ventroposterior nucleus was all the more
the extensive flattened tissue sections, large composite image files remarkable because the tracer deposit covered the whole S1
weremadefrom300to600individualimages(600dpigrayscaleJPEG) area. Thalamic labeling in the 2 additional experiments with D
using Analysis and Canvas software. By zooming into the images, we o
extensivelayer1depositsinlateralanddorsalneocorticalareas w
drewlabeledaxonsinlayer1;axonsindeepertissuezones(revealedby n
thepresenceofcellsomatabythethionincounterstain)wereignored. (Supplementary Fig. SM1 and Table SM1) confirmed these loa
Because of the impossibility of resolving branching points from observations. de
iwneterresedcrtaiownnsinasaxcoonnsairsbtionrgsaotfthinisdempaegnndifiecnattioseng,mlaebnetlse.dTaxhoenmaribdodrles reaScmhaeldl breytrthogeraladregetrdaecpeorsitdsepyioeslidtedinlalbaeyleinrg1ininmaanryeaosf nthoet d from
cerebral artery and its branches, as well as tissue landmarks such as thalamicnucleialreadylabeledbythelargedeposits,butalsoin h
therhinalsulcusorlateralolfactorytract,wereusedasreferencesfor some additional nuclei. For example, following an insular area ttp
aosvseermtbhleinwghtohleehseecmtiiospnhseirnet.oTahepasunporearmioirccveireewbraolfvtehineslawbeerleedusaexdonass deposit(Fig.3A),labeledcellsappearedinthedorsomedialand s://a
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reference for locating the dorsolateral/medial watershed of the centralis medialis nuclei; likewise, a deposit in the medial ad
hemisphere. frontal cortex (Fig. 3B) labeled cells in the dorsomedial em
The hemispheres processed ‘‘in toto’’ to reveal BDA were kept nucleus;andadepositintheretrosplenialcortexlabeledmany ic
floatingin1%sodiumazideinPBat4(cid:2)C.Forexamination,theywere cellsinthelateraldorsalaswellastheanteriornuclei(Fig.3H). .ou
p
transferred toa PB-filledchambermountedon themicroscope stage, Single small tracer deposits situated in different locations .c
flattenedunderacoverslip,transilluminated,andexaminedunderthe within the same dorsolateral cortical zone already explored om
103 objective. Mosaic images were generated and the labeling /c
with the extensive tracer deposit experiments showed fewer e
reconstructed as above. In this unsectioned, non-defattened, and rc
wwonaeltryeera-vmfirsaoicbutlnieot.endMooftritsehsoueveel,arb,oenblleeydcatauhxseoenlasobfceonleuodlnddabexehoyncdasrpanttueeraderdttiihsnseuaepsiiatnhlgilcseukrfnofeaccsasel, lpcaoabtnetseleirsdntescnoetfl(lscceotlmhlapdnaisraterfitbFeiurgtstioh2neawenxditthe3inCns--itGvhe)e.dtehpaolasmitsu;shwoewreevsetrr,ictthley or/article
plane at a microscope magnification sufficient for axon visualization. The combined evidence from all the layer 1 deposits, large -a
Tofhethseeflaabcetolerdsparxoobnasbilnyclaoynetrri1buintetdhetoseaecxopnesriidmeeranbtsle. underestimation and small, shows that the M-type thalamocortical cell pop- bstra
ulationisalargeoneandiswidelydistributedinthethalamus. c
Althoughourexperimentsdidnotaddresstheproportionthat t/19
VGluT2Immunohistochemistry M-typecellsrepresentoverthetotalneuronsofeachthalamic /10
Tadodimtioasnsallarbeefelrtehnaclaemcoocmoprtairciasolnsywnaitphticousriteexspienritmheenntse,owceoritmexmuasnoasn- nuclei,thelargenumberoflabeledcellsinsomenucleistrongly /238
tainedsometissuesectionsforVGluT2.Coronalcorticalsectionsfrom3 suggests that in them M-type cells form the majority. In 0/6
animalswereimmunolabeledwithanaffinity-purifiedrabbitanti-VGlut2 contrast, some thalamic nuclei such as the ventral posterior 0
0
serum(1:500Chemicon,Temecula,CA;Fujiyamaetal.2001).Labeling nuclearcomplex,theventraldivisionofthemedialgeniculate, 17
wasvisualizedvialinkagetoabiotinylatedgoatanti-rabbitserum(1:100 the ventral--medial portions of the dorsal lateral geniculate, 3 b
Chemicon) following a standard avidin--biotin--peroxidase staining submedius, anterodorsal, and anteroventral nuclei remained y g
protocolwithDABaschromogen.Parallelsectionsinwhichtheprimary u
largely free oflabeled cells inevery experiment. e
antiserumwassubstitutedbynormalrabbitserumyieldednolabeling. s
t o
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Results ThalamicInput to Layer 1Is a SpecificCombination of 6 A
Afferents fromMany Nuclei p
ManyThalamic Nuclei Contain LargeNumbers ofLayer Visual comparison of neuron distribution in experiments ril 2
0
1--Projecting Thalamocortical Neurons involving small deposits in different cortical areas (Fig. 3) 19
AlargefractionoftheM-typecellpopulationwasvisualizedby indicated that the combination of thalamocortical inputs
meansofretrogradetracerexperimentsthatcreatedadeposit reachinglayer1variessubstantiallybetweenareas.Foradirect
that was very extensive but remained confined to layer 1. comparisonthatwouldbeunaffectedbydifferencesinabsolute
In addition, a large series of small tracer deposits were used neuron number between experiments, we compared the
to analyze in detail the spatial distribution of M-type inputs percentage of neurons labeled in each nucleus to the total
within the zones sampled by the large deposits as well as to number of labeled cells in the thalamus (Fig. 4). This analysis
probe M-type input to areas not reached by the extensive clearly shows that thalamic input reaching layer 1 in each
deposits(Fig. 1, seealso Supplementary Fig.SM1). cortical area is a combination, in specific proportions, of
Atypicalextensivelayer1depositisillustratedinFigs1F--G afferents from awide array of nuclei.
and 2. In this experiment, the tracer-impregnated layer 1 in In addition, the comparison of the thalamic patterns of
most of the lateral frontal and parietal cortices as well as labeling in our series of small deposit experiments revealed
sectorsofthetemporalandoccipitalcortices.Massivenumbers marked differences in the degree of divergence along the
of cells were labeled in the ventrolateral, ventromedial, cortical surface of M-type pathways originating in different
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Figure2. Thelayer1--projectingthalamocorticalcellsarealargepopulationspreadthroughoutthethalamus.(A--H)Linedrawingsofrepresentativecoronalthalamicsections. t/1
Eachblackdotrepresentsonelabeledcellsoma.Fiberbundlessuchasthemamillothalamictract,thestriamedularis,theretroflexbundle,orthemediallemniscusareshadedin 9/1
gray.NotethatalthoughthetracerdepositcoveredmostofareaS1,theventroposteriornucleus(VP)islargelyunlabeled.(A#,B#,F#,G#)Epifluorescenceimagesofthelabeling 0
intherectanglesin,respectively,panels(A,B,F,andG).SeeTable1forabbreviations.Furtherdetailsofthelabeledcellscanbeappreciatedbyzoomingintotheimages.Ahigh- /23
magnificationepifluorescenceimageoflabeledcellsisshowninSupplementaryFigureSM3.Bars:(A--H)51mm;(A#--B#),(G#--H#)5500lm. 80
/6
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nuclei (Figs 3 and 4). For example, M-type neurons in nuclei deposits were few, relatively small, and placed without 3 b
such as the dorsomedial, anterodorsal, or dorsal lateral aspecificpattern,theimplicationseemstobethatthenumber y g
u
geniculatewerelabeledonlybylayer1depositsinaparticular of cells in these nuclei that branched their axons into widely e
s
cortical field, indicating a limited tangential spread of their separated targetareas issubstantial. t o
n
axons.In contrast,nuclei such astheventromedial, thelateral Ontheotherhand,eveninthenucleiwithaxonsdiverging 0
6
posteromedial, or the posterior nucleus were labeled from widely in the cortex, the position of cells labeled in different A
dtheepiorsaitxsonloscdativeedrgineabrvoaardielytyinofthceorctoicrtaelxa.reas, suggesting that edxeppoersiimtseonntsmreevdeiaallsfraocnrtuadlearteoapsolgabraeplehdicdaloorsradlepr.oFrotironexsaomfpthlee, pril 2
0
Innervation of wide cortical areas from the latter nuclei ventromedial nucleus (Fig. 3A), whereas deposits on parietal 19
could result from a widespread dispersal of individual axons, areas labeled more caudolateral portions of this nucleus (Fig.
each innervating a single cortical zone, or from the divergent 3D). Thus, even if most cells in the ventromedial nucleus
branching of single axons to reach multiple zones. To branch their axons toward multiple cortical areas, there still
distinguish between these 2 possibilities at the population seems to beacrude topographic order tothese pathways.
level,weplacedepipialdepositsofFBorDYindistantregions
ofthecerebralhemisphereinalimitednumberofexperiments Large NumbersofDifferent ThalamicAxons Converge in
(n = 5; experiments numbered with ‘‘a’’ or ‘‘b’’ suffixes in Every Point ofLayer1
SupplementaryTableSM1)andthenlookedfordouble-labeled Astrikingobservationfollowingsmalllayer1depositswasthat,
neurons in the thalamus (Supplementary Figs SM2 and even when the impregnated layer 1 area was very small,
SM3D--E). Double-labeled cells were found in only a handful numbersoflabeledthalamiccellswereconsistentlyhigh(Fig.5).
of thalamic nuclei, and interestingly, these nuclei (highlighted Moreover, this observation was repeated in every cortical area
byasterisksinthetableinFig.4)werepreciselythoseshowing explored.TheimplicationisthatlargenumbersofM-typeaxons
the widest tangential axon dispersal. Because our double convergewithineverysmallregionoflayer1.
2384 ThalamocorticalInputtoApicalDendriticTufts d Rubio-Garridoetal.
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Feaigcuhroef3w.hiIcnherveecreyivceodrtaicasmlaarlelala,ysemra1lldterpaocesirtdinepaodsiiftfseriennltayceorrt1icablaacrkeala.bTehlamlaumltiicplleabtehlainlagmisicsnhuocwleni.in(A6--cI)oCroonmalpsaericstoionnosfatrhreantgheadlafmroicmlarobsetlirnagl(ilnef9t)rteopcreasuednatla(triivgehte).xEpaecrihmbelnatcsk, ril 20
dotrepresentsasinglecell.Notethatsomenuclei,liketheventromedial(VM),anteromedial(AM),orlateralposteriormedialrostralpart(LPMR),containedlabeledcellsinmost 19
orvirtuallyallcorticalregionsfollowingthelayer1deposit.Incontrast,nucleisuchasthedorsallateralgeniculate(DLG,rowF)orthemedialgeniculate(MGD,rowI)were
labeledonlybylayer1depositsinaparticulararea.SeeTable1forotherabbreviations.Bar51mm.
The methods employed do not allow a precise quantitative injection in brain tissue is difficult to determine (Mesulam
assessment of the number of thalamic neurons converging in 1982).However,inourepipialdeposits,dendriticbacklabeling
anygiven regionoflayer1;however, asanapproximation, we ofpyramidalcellsinlayer5providedareliableinternalcontrol
counted, for each case, all the labeled neurons in a series of for the zone from which tracer transport actually had taken
thalamus sections (1:5 sections, each 40 lm thick) and place. Because such back labeling consistently matched the
compared the result with the area of layer 1 surface covered tracer-impregnated surface in layer 1 (Fig. 1B,C,E,F), we are
by the corresponding cortical deposit reconstructed from the confident that the latter closely corresponds to the area from
serial cortical sections (Fig. 4). The zone from which tracer whichtracertransportoccurredineachexperiment.Inaddition,
uptake and transport effectively occurs following a tracer to compensate for the shrinkage in surface of the cerebral
CerebralCortexOctober2009,V19N10 2385
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Figure4. SemiquantitativeanalysisofthedistributionofM-typethalamocorticalcellsprojectingtodifferentcorticalregions.Upperleftcorner:flatcortexmapshowing12small stra
epipial depositexperiments,each ina different corticalregion.To normalizeandcompareacross theseexperiments,the percentages that thecells labeledin eachnuclei c
representoverthetotalforthethalamiccellslabeledineachexperimentaredisplayedaslevelsofgrayinthetableattheleft.Inthetable,nucleiarelistedaccordingtobroad t/1
9
functionalaffinities.Theexperimentnumberisindicatedatopeachcolumn.Thisanalysisconfirmstheimpressiongainedfromtheobservationofindividualthalamicsectionsthat /1
somenucleidispersetheiraxonsovervastswathsoflayer1,whereasothernucleiinnervatelayer1inafeworinevenonlyonearea.SeeTable1forabbreviations. 0
/2
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hemisphereobservedbetweenthefreshratcortextissueandthe morelimitedcorticalareas.Todirectlyobserveandcomparethe 0
0
fixed, sucrose-embedded, and dry-mounted cortical sections, actual distributionand morphologyof theseaxons withinlayer 1
7
3
a correction factor of 1.35 (Avendan˜o C, personal communica- 1, we made small BDA injections in some of these thalamic b
y
tion)wasappliedtothemeasuredsurface.Thisanalysisrevealed nucleiandexaminedthelabelinginthecortex(TableSM1). g
ameandensityratioof1025(±250.5standarddeviation)labeled In serial coronal sections (Supplementary Fig. SM4) of ue
s
neurons in the thalamus per square millimeter of fresh layer 1 brainsthatreceivedsmallinjectionsinsomeofthenucleithat t o
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surface. As cells were counted in only one fifth of the total innervatewideregionsoflayer1,suchastheventromedialor 0
6
thalamictissue,aconservativeestimatewouldthen bethatthe themediallateralposteriornucleus,thalamicaxonswereseen A
nsquumarbeermoiflldimiffeetreernotfMl-itvyinpgetrhaatlalamyeicrn1eumruosntsbreeacinhitnhgearnaynggieveonf esunbtecroirntgictahlewcheirteebrmalahtteemr.isApthemreulatnipdleexptoeinndtisn,gistohlraoteudghptrhoe- pril 2
0
4000--5000.Notethat,withoutcorrectionforsurfaceshrinkage, cesses extended radially across the cortical layers, and in 19
thisdensitywouldbeevenhigher. occasional favorable sections, they could be followed all the
Finally, neuron numbers in experiments situated in very way up to layer 1. Many of these processes arborized also in
different points of the cerebral surface, including primary layers5or3.Withinlayer1,theyremainedconfinedtoathin
sensory, motor, association, or limbic areas, were compared stratum (~70 lm deep) in the uppermost half of layer 1
withtheextentofeachdeposit.Thisanalysisrevealedthatthe (sublayer 1a, Vogt 1991) as essentially flat and tangentially
massive spatial convergence of thalamic axons in layer 1 is oriented axonal arbors. For this reason, coronal sections are
quitesimilar throughout thecortex (R2=0.93; Fig.6). clearly inadequate for studying global morphology of these
axon arbors. Thus, we examined the axon labeling in layer 1
Distribution in Layer 1ofTangential M-Type Axon either directly on whole-mounted unsectioned and flattened
Arborizations hemispheres or in sections parallel to the pial surface taken
Retrogradetracerdepositsinlayer1seemedtoindicatethatM- fromflattenedhemispheres.Fromtheseimages,wegenerated
type neurons in some nuclei distribute their axons over wide panoramic views of the labeling on the cerebral hemisphere
areas of the cortex, whereas those in other nuclei innervate surface (Fig. 7).
2386 ThalamocorticalInputtoApicalDendriticTufts d Rubio-Garridoetal.
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Figure5. Asmalltracerdepositinlayer1labelsalargenumberofneuronsinthethalamus.(A)Extentofatypicalsmalldepositreconstructedonacorticalsurfacemap.Inthis 0
0
particularexperiment(Case20,seeFig.1A--B),theFBdepositimpregnated1.8mm2ofcorticallayer1.(B--F)Panoramicepifluorescenceimagesshowingretrogradelylabeled 1
7
neuronsomatainthethalamus.Theimagescorrespondtotheregionsindicatedbyrectanglesonthelinedrawingsinpanel(G).Furtherdetailsofthelabeledcellscanbe 3
b
appreciatedbyzoomingintotheimages,aswellasinSupplementaryFigureSM3.(G)Linedrawingsfromanentireseriesofsections(1outof5,40-lm-thickeach,spacedby y
200lmapart).Sectionsarearrangedfromrostral(upperleftcorner)tocaudal(lowerrightcorner).Eachdotrepresentsasinglecell.Thetotalnumberofneuronslabeledinthis gu
particularseriesofthalamicsectionswas2211.SeeTable1forabbreviations.Bar:(A)52mm;(B--F)5500lm;(G)51mm. es
t o
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0
6
In these panoramic views, the extent and number of the consistently produced a single relatively small zone of layer 1 A
aexviodnenat.rbFoorrselxaabmelpelde,bfoyllosowmineg samsamllaltlh(a<la1m50iclimnjedcitaimonesteri)s axonlabeling within theprimaryvisual cortex(Fig. 7D--G). pril 2
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BDA injection in the ventromedial nucleus, one of the nuclei 19
notedintheretrogradeexperiments tohavea hightangential FineMorphology ofThalamocortical AxonArbors in
dispersal of its axons (see also Herkenham 1979; Monconduit Layer1
and Villanueva 2005) also exhibited numerous subpial axon Even with our smallest BDA injections in the thalamus, the
arbors extending over most of the rostral half of the cerebral overlap and high number of labeled layer 1 axon arbors made
hemisphere(Fig.7B--C).Thesesubpialarborsweredistributed individual axon reconstructions impossible. Nevertheless, at
very unevenly, accumulating in several ‘‘patches’’ and leaving high magnification, some morphological features could be
adjacent zones almost unlabeled. Likewise, a slightly larger readily appreciated. Near the point of entry from the un-
injection near the ventral and lateral border of the ventrome- derlying layers into layer 1, many axons made a sharp turn,
dial nucleus produced even more extensive labeling (Supple- taking a tangential trajectory. Most arbors ramified frequently
mentary Fig. SM5). In contrast, comparable BDA injections within sublayer 1a (Fig. 8C--F). In most sites, multiple arbors
placedinothernucleisuchasthedorsallateralgeniculate(one were overlapping, their branches forming a crisscrossing
of the nuclei identified in the retrograde labeling analysis as subpial mesh (Fig. 8B--D). Some few arbors consisted of long,
givingrisetoM-typeprojectionswithlowtangentialdispersal) sparingly branched axon segments that followed roughly
CerebralCortexOctober2009,V19N10 2387
given spot of layer 1; 3) within layer 1, M-type axon arbors
extend as a patchy tangential mesh of crisscrossing branches
with frequent bouton-like swellings; and 4) M-type pathways
are not always ‘‘diffuse’’: the axons from some nuclei diverge
widely, covering large swaths of the cerebral hemisphere,
whereas those from other nuclei arborize within only a single
cortical area.
The M-Type Cell Population Is LargeandWidespread
Because the intensively studied sensory thalamic nuclei
(ventroposterior,lateralgeniculate,andmedialgeniculate)are
made predominantly of C-type cells, the anatomy, functional D
o
properties,andsynapticrelationshipswiththecortexofthese w
n
cells have been worked out in considerable detail (reviews in lo
a
Castro-AlamancosandConnors1997;ShermanandGuillery2005; d
e
Jroenceeisve2d00m7u).chInlescsonattrtaesntt,ioMn-t(yLpoerenthtealadmeoNcoort1ic9a3l8;ceKllisllachkaevey d from
andEbner1973;FrostandCaviness1980;Herkenham1986).As h
aresult,dataonM-typethalamocorticalpathwaysarescantand ttp
s
fragmentary, and considerable uncertainty remains about their ://a
c
anatomical weight, thalamic origin, and function (Llina´s et al. a
d
Figure 6. Approximate estimation of the number of cells innervating 1 mm2 of 2002;Kubotaetal.2007;Grazianoetal.2008). em
cortical layer 1. Surface area covered by the tracer deposits in each of the 12 Some years ago, it was hypothesized that these cells might ic
.o
experiments shown in Figure 4A after correction for cortical surface shrinkage actuallybeabroadneuronclassthatwasspreadwidelyacross u
p
(Avenda˜no C, personal communication) was plotted against the total number of thethalamusandonethatwasfundamentallydifferentfromthe .c
retrogradely labeled thalamic neurons counted in a whole rostrocaudal series of thalamocorticalneuronstargetingthemiddlelayers(Avendan˜o om
thalamussections(1:5sections).Thisdensityratioprovidesadirectestimateofhow /c
mmamn2yotfhlaalyaemro1c.oTrhtiecamlecaenllsdeenxtseitnydraattioleisas1t0s2o5m±ep2a5r0tcoeflltshepierramxomn2.inNtootaen,yhogwiveevner1, eotbsaelr.v1e9d9t0o;rJeocneeisve1d9i9ff8e)r,eanstienapcuhtsoafntdh/eor2hnaveeurdoifnfetryepnetssowmaas erco
tn5hu0am0t0b.ceerRlloesfgwrtehesarsleaiomnciocuacnnetaelllysdsiinisnnesorhnvoalywtinsagafi1ftrmhemmof2artokhfaeblalyytoetrsai1ml tmilhaaurlsatdmebinecsoittniysstiuhnee,eosvrdoeeryrthoesfe4na0cs0otur0ya--,l s2i0z0e1s)a.nMdo/oreroevxepr,rethsseidricffoerrteicnatlctaalrcgieutmss-beienmdiendgcpormotpelienmse(nJotanreys, r/article
association,motor,orlimbiccorticalareaexamined(R250.93). asthalamicaxonsarborizingincorticallayer1donotarborize -ab
in layer 4 (despite often doing so in layers 5a and/or 3b), stra
whereas axons arborizing in layer 4 do not arborize in layer 1 c
straight trajectories without an apparent preferred direction. (Boyd and Matsubara 1996; Catalano et al. 1996; Descheˆnes t/19
The longest of such segments rarely exceeded 2 mm. This etal.1998;Gheorghitaetal.2006;Casagrandeetal.2007).Our /10
apparent limitation in maximum length was the same in both finding that M-type neurons are present in many thalamic /23
sectioned and whole-mounted tissue. 8
nucleiseemsconsistentwiththematrixhypothesis,aswellas 0
Axonarborsinlayer1showednumerousirregularlyspaced /6
with observations following anterograde tracer injections in 0
swellings, suggestive of en passant boutons (Fig. 8E--F). individualratnuclei(KrettekandPrice1977;Herkenham1986; 01
7
Between swellings, axon caliber was variable, but always very 3
Berendse and Groenewegen 1991; Van Groen and Wyss 1995; b
small(<0.5lm).Interboutondistancesweremeasuredinlarge y
Linke and Schwegler 2000; Kimura et al. 2003; Gauriau and g
samples of axon segments labeled from the ventromedial u
Bernard 2004; Vertes et al. 2006) or superficial retrograde e
nucleus, lateral posterior, and the dorsal lateral geniculate tracer deposits in a single area (Parnavelas et al. 1981; Rieck st o
nucleus. No correction was applied for shrinkage in these n
and Carey1985; Mitchell and Cauller 2001). 0
measurements. Mean interspace bouton distances and their 6
We demonstrate here that M-type neurons are a vast cell A
rdaifnfgeeresnotfnvuarcilaetiio(n5.w23erelmsimfoilrarvbeenttwroemenedaixalo;n5s.2o3rigfionratliantgerianl pthoaplaumlautiso.nThespserecaedllswariedeplryevaanledntuinnethveenvleyntrtohmroeudgihaol,uvtenttrhael pril 2
posterior medial rostral part; and 4.3 for DLG [dorsal lateral anterior, and interanteromedial nuclei, as well as in large 019
geniculate nucleus] axons; Fig. 8H). Comparison with pub-
portionsoftheventrolateral,posterior,lateralposterior,dorsal
lished measurements in typical C-type axons (3.59 lm in
lateral geniculate, parafascicular, and medial geniculate com-
ventroposterior nucleus axons innervating layer 4 ‘‘barrels’’ in
plex(Fig.9A).Thesubmedius,anterodorsal,anteroventral,and
the rat somatosensory cortex [Lu and Lin 1993]) suggests
portions of the laterodorsal, dorsomedial, and reuniens nuclei
a comparable abundanceof boutons per axon length unit.
were scarcely labeled; however, they innervate layer 1 pro-
fusely in medial and lateral limbic areas not reached by our
deposits (Krettek and Price 1977; Herkenham 1986; Shibata
Discussion
1993;VanGroenandWyss1995;Vertesetal.2006),andsowe
We mapped the thalamic origin and cortical distribution of assumethatthesenucleiwouldalsocontainnumerousM-type
thalamocortical pathways innervating cortical layer 1 (M-type cells. In contrast, unlabeled portions of the lateral and medial
neurons). The main findings are that 1) M-type neurons are geniculate,ventrallateralnuclei,andthewholeventroposterior
numerous in many thalamic nuclei; 2) axons from large nucleus probably lack M-type neurons: we extensively probed
numbers of M-type thalamocortical neurons converge at any their target areas with retrograde tracers and found nothing,
2388 ThalamocorticalInputtoApicalDendriticTufts d Rubio-Garridoetal.
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Figure7. TangentialspreadinthecortexvariesgreatlybetweenM-typeaxonsoriginatedindifferentthalamicnuclei.(A)Lateralviewoftherat’sleftcerebralhemisphere.For /6
0
reference,thepositionofmiddlecerebralartery(MCA)anditsmainbranchesareindicatedbydashedlines.Comparewithpanels(C)and(E).(B)Coronalsectionshowingthe 0
1
centerofasmalliontophoreticdepositofBDA(arrow)intheventromedial(VM)thalamicnucleus.CyOcounterstain.Thisdepositbackfilled~150ventromedialneuronsomata.(C) 7
3
Reconstruction ofthalamocortical axonarbors labeled inlayer 1 afterthe injectioninpanel (B) asobserved ona flattened whole mountofthe cerebralhemisphere.MCA is b
y
representedbythickdashedgraylines.Thalamocorticalaxonsinlayer1arethickened~100timestomakethemvisibleatthisscale.Notethatalargeswathofthecerebralsurfaceis g
reachedbytheaxonsarisinginthesmallthalamiczoneinjectedwithBDA.Forcomparison,the‘‘barrel’’layer4domainsintheposteromedialbarrelsubfield(PMBSF)asrevealedby ue
wCyitOhinhisthtoechareemaisotfry1aorre2albsaorrsehlodwomn.aIitnsis(tJoenbseernemanedmKbiellarecdketyha1t9C87-t;yApernaoxldonestaalr.is2in0g01in).t(hDe)vSemntarlolpioonsttoeprihoorrmeteicdidaelpnouscilteousfB(VDPAM(,arproanwe)liAn)thheavmeobsetemnesdhioawlpnarttooafrbthoerizdeoressaslelnattiearlalyl st on
geniculatenucleus.Thionincounterstain.(E)Thalamocorticalaxonarborslabeledinlayer1intheexperimentshowninpanel(D)reconstructedfromserialtangentialsectionsof 0
6
aflattenedhemisphere.(F--G)FollowingalargeiontophoreticBDAdepositaboutthesamelocationasthepreviouscase(panelD),butinvolvinginthiscasemanythousandsof A
thalamiccells,labellayer1axonsonlywithintheprimaryvisualarea.Reconstructionfromserialtangentialsections.Bar:(A,B,D,F)52mm;(C,E,G)5200lm. pril 2
0
whereasanterogradestudiesbyotherssuggestaC-typeneuron axons to reach 2 areas. Moreover, the patchy appearance 19
predominance (Herkenham 1986; Arbuthnott et al. 1990; resulting from the multiple small subpial arbors in a wide
Kageyama and Robertson 1993; Kimura et al. 2003). Likewise, region of cortical labeling (Fig. 7) is consistent with M-type
we interpret the scant labeling in nuclei like the anterior and cells in these nuclei having a ‘‘widespread’’ axon that gives off
posterior intralaminar, paraventricular, intermediodorsal, and multiple branches in the subcortical white matter (Fig. 9C;
peripeduncularnucleiasanindicationthattheyharborfewM- Descheˆnes et al. 1998; Galazo et al. 2008). In contrast, other
type cells because their main targets seem to be the deep thalamic nuclei were labeled from only a single cortical area,
cortical layers, the striatum, and/or amygdala (Herkenham indicatingthattheirM-typeneuronshaveafarmore‘‘localized’’
1986;BerendseandGroenewegen1991;Descheˆnesetal.1996; axondistribution.Single-axonstudieshaveinfactshownsome
Linke andSchwegler 2000;Vertes and Hoover 2008). M-typeaxonsarborizing within asingle area (Descheˆnes et al.
Nuclei such as the ventromedial or posterior were labeled 1998; Casagrande et al. 2007). Overall, the M-type cell class
from widely separate cortical areas. The double-labeling thus seems to show substantial variation regarding 1) axon
experiments(seealsoMonconduitandVillanueva2005)show branching to one or multiple areas (widespread or localized
thatatleastsomeM-typeneuronsinthesenucleibranchtheir axons);2)arborizationintocorticallayersinadditiontolayer1
CerebralCortexOctober2009,V19N10 2389