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Brainstem Projections to Midline and

Intralaminar Thalamic Nuclei of The RatKARL E. KROUT, REBECCA E. BELZER, AND ARTHUR D. LOEWY*

Department of Anatomy and Neurobiology, Washington University School of Medicine,St. Louis, Missouri 63110

ABSTRACTThe projections from the brainstem to the midline and intralaminar thalamic nuclei were

examined in the rat. Stereotaxic injections of the retrograde tracer cholera toxin -subunit(CTb) were made in each of the intralaminar nuclei of the dorsal thalamus: the lateral

parafascicular, medial parafascicular, central lateral, paracentral, oval paracentral, andcentral medial nuclei; in the midline thalamic nucleithe paraventricular, intermediodorsal,mediodorsal, paratenial, rhomboid, reuniens, and submedius nuclei; and, in the anteroven-tral, parvicellular part of the ventral posterior, and caudal ventral medial nuclei. Theretrograde cell body labeling pattern within the brainstem nuclei was then analyzed. Nearlyevery thalamic site received a projection from the deep mesencephalic reticular, pedunculo-pontine tegmental, dorsal raphe, median raphe, laterodorsal tegmental, and locus coeruleusnuclei. Most intralaminar thalamic sites were also innervated by unique combinations ofmedullary and pontine reticular formation nuclei such as the subnucleus reticularis dorsalis,gigantocellular, dorsal paragigantocellular, lateral, parvicellular, caudal pontine, ventralpontine, and oral pontine reticular nuclei; the dorsomedial tegmental, subpeduncular teg-mental, and ventral tegmental areas; and, the central tegmental field. In addition, mostintralaminar injections resulted in retrograde cell body labeling in the substantia nigra,nucleus Darkschewitsch, interstitial nucleus of Cajal, and cuneiform nucleus. Details con-cerning the pathways from the spinal trigeminal, nucleus tractus solitarius, raphe magnus,

raphe pallidus, and the rostral and caudal linear raphe nuclei to subsets of midline andintralaminar thalamic sites are discussed in the text. The discussion focuses on brainstemthalamic pathways that are likely involved in arousal, somatosensory, and visceral functions.J. Comp. Neurol. 448:53101, 2002. 2002 Wiley-Liss, Inc.

Indexing terms: attention; autonomic nervous system; cerebral cortex; nociception; sleep;vigilance

The midline and intralaminar thalamic nuclei havebeen called nonspecific relay nuclei, because they werethought to project to vast areas of the cerebral cortex (e.g.,Lorente de No, 1938; Morison and Dempsey, 1942; Schei-

bel and Scheibel, 1972; Jones and Leavitt, 1974). How-ever, this idea has been refuted because each of the mid-line and intralaminar thalamic nuclei have beendemonstrated to project to limited and specific corticalregions, as well as to subregions of the basal ganglia andamygdala (e.g., Berendse and Groenewegen, 1990, 1991;Turner and Herkenham, 1991; Yasui et al., 1991; for re-

view, see Bentivoglio et al., 1991; Groenewegen and Be-rendse, 1994). These forebrain circuits affect behavioral orcognitive responses and may be modulated by ascendingafferent information arising from the brainstem and spi-nal cord (Alexander et al., 1990; Groenewegen et al., 1990,1999).

A classic example of this brainstem regulation of fore-brain activity was demonstrated by Moruzzi and Magounin 1949. They found that electrical stimulation of thebrainstem reticular formation caused desynchronization

of the cortical electroencephalogram (EEG). They sug-gested that these profound cerebral cortical changes were

Grant sponsor: National Institute of Heart, Lung, and Blood of theNational Institute of Health; Grant number: HL-25449.

*Correspondence to: Arthur D. Loewy, Department of Anatomy andNeurobiology, Box 8108, Washington University School of Medicine, 660 S.Euclid Avenue, St. Louis, MO 63110. E-mail: [email protected]

Received 29 November 2001; Revised 28 January 2002; Accepted 31January 2002

DOI 10.1002/cne.10236

Published online the week of April 29, 2002 in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 448:53101 (2002)

2002 WILEY-LISS, INC.

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due to activation of a group of neurons lying in the core ofthe reticular formation and proposed that this ascendingreticular activating system (ARAS) exerted a major influ-ence on sleep, wakefulness, and arousal (for review, seeGottesmann, 1999; Vincent, 2000). Subsequent anatomicand physiological studies have shown that the pathwaysresponsible for these effects originate from the brainstemreticular formation and terminate within the midline andintralaminar thalamic nuclei (e.g., Morison and Dempsey,1942; Hunter and Jasper, 1949; Nauta and Kuypers, 1958;Scheibel and Scheibel, 1958; Bowsher, 1975). In the 50years since this discovery, the exact cells of origin thatinnervate the individual midline and intralaminar tha-lamic nuclei have not yet been determined.

Numerous anterograde studies have examined thebrainstem inputs to the thalamus (e.g., Peschanski and

Besson, 1984c; Jones and Yang, 1985; Vertes et al., 1986,1999; Hallanger and Wainer, 1988; Bernard et al., 1990;

Vertes, 1991), but none of them have systematically de-fined the full extent of brainstem sites which innervatethe midline and intralaminar thalamic nuclei. Similarly,several retrograde studies have been published which de-scribed the brainstem inputs to these thalamic targets,but these either relied on injections which encompassedmultiple thalamic nuclei or limited the scope of their in-

vestigation to only one or two thalamic nuclei (e.g., Hal-langer et al., 1987; Cornwall and Phillipson, 1988; Groe-newegen, 1988; Carstens et al., 1990; Niimi et al., 1990;

Yoshida et al., 1992; Bolton et al., 1993; Newman andGinsberg, 1994). Therefore, a mapping of the completebrainstem inputs to individual midline and intralaminarthalamic nuclei has not yet been described and would be

Abbreviations

A5 A5 area

AD anterodorsal thalamic nucleus AM anteromedial thalamic nucleus AP area postrema APT anterior pretectal nucleus AV anteroventral thalamic nucleusC L ce nt ra l l at er al t ha la mi c nu cl eu sCLiR caudal linear nucleus of the rapheCM central medial thalamic nucleusCnF cuneiform nucleusCu cuneate nucleusDk Nucleus of DarkschewitschDmTg,DMTg dorsomedial tegmental areaDPGi dorsal paragigantocellular nucleusDpMe deep mesencephalic reticular nucleusDR dorsal raphe nucleusDRc dorsal raphe nucleus, caudal partDRd dorsal raphe nucleus, dorsal partDRv dorsal raphe nucleus, ventral part

DRvl dorsal raphe nucleus, ventrolateral partECIC external cortex of the inferior colliculusf fornixfr fasciculus retroflexusGi gigantocellular reticular nucleusGiA gigantocellular reticular nucleus, alpha partGr gracile nucleusIC inferior colliculusIMD intermediodorsal thalamic nucleusINC interstitial nucleus of CajalIP interpeduncular nucleusIRt intermediate reticular nucleusLC locus coeruleusL D la te ro do rs al th al ami c nu cl eu sLDTg laterodorsal tegmental nucleusLGN lateral geniculate nucleusLH lateral hypothalamic areaL Hb la te ra l ha be nu la r nu cl eu sL M la te ra l ma mmi ll ar y nu cl eu s

LP lateral posterior thalamic nucleuslPF lateral parafascicular thalamic nucleusL Rt la te ra l r et ic ul ar nuc leusMD med io do rs al t ha lami c nu cl eu sMdD medullary reticular nucleus, dorsal partMdV medullary reticular nucleus, ventral partMG med ia l ge ni cul at e nuc leu sMH b med ia l ha be nu la r nu cl eu sMM med ia l ma mmil lar y nu cl eu smPF medial parafascicular thalamic nucleusMR median raphe nucleusN TS nuc leus of t he s ol ita ry t ractOPC oval paracentral thalamic nucleusPAG periaqueductal gray matterPaR pararubral nucleusPB parabrachial nucleusPBP parabrachial pigmented nucleus

pc posterior commissure

P C par ac en tr al th al ami c nu cl eu sPC-c paracentral thalamic nucleus, caudal partPC-r paracentral thalamic nucleus, rostral partPCRt parvicellular reticular nucleusPnC pontine reticular nucleus, caudal partPnO pontine reticular nucleus, oral partPnV pontine reticular nucleus, ventral partP o pos ter io r th al am ic n uc le ar g ro upPPTg pedunculopontine tegmental nucleusPr prepositus nucleusPr5 principal sensory trigeminal nucleusP RC pre co mmi ss ur al n uc le usP T par at en ia l t ha la mi c nuc leu sPVA paraventricular thalamic nucleus, anteriorPVP paraventricular thalamic nucleus, posteriorPVT paraventricular thalamic nucleus, middleRe reuniens thalamic nucleusRh rhomboid thalamic nucleus

R IP rap he i nt erpo si tus n ucl eusRLiR rostral linear nucleus of the rapheRMg raphe magnus nucleusRN red nucleusRPall raphe pallidus nucleusRRF retrorubral fieldRt reticular thalamic nucleusRVLM rostral ventrolateral medullaSC superior colliculuss cp sup er io r c er eb el la r pe du nc les m st ri a me dul lar is o f t he t ha la musS M sub med ius t hal amic nuc le usSNpc substantia nigra, compact partSNpr substantia nigra, reticular partS p5 spi na l t ri gemi na l nu cl eu sSp5C spinal trigeminal nucleus, caudal partSp5I spinal trigeminal nucleus, interpolar partSp5O spinal trigeminal nucleus, oral part

SPTg subpeduncular tegmental nucleusSRD dorsal reticular subnucleus (Subnucleus reticularisdorsalis)

S ubC sub co er ul eu s nu cl eu sS uM sup ra mamm il lar y nuc leusTMv tuberomammillary nucleus, ventral VA ventral anterior thalamic nucleus Ve vestibular nuclei VL ventrolateral thalamic nucleus VM ventromedial thalamic nucleus VMc ventromedial thalamic nucleus, caudal part VP ventral posterior thalamic nucleus VPM ventral posteromedial thalamic nucleus VPpc ventral posterior thalamic nucleus, parvicellular part VTA ven tr al te gme nt al ar eaxscp decussation of the superior cerebellar peduncleZI zona incerta

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particularly useful in terms of defining which regions ofthe brainstem could possibly influence the basal gangliaand cerebral cortex. In the present study, we made highlyrestricted injections of the retrograde tracer cholera toxin-subunit (CTb) in each midline or intralaminar thalamicnucleus and provided maps of the retrograde cell bodylabeling throughout the entire brainstem. These data willcomplement earlier reports in this series on the thalamicafferents from the periaqueductal gray (Krout and Loewy,2000b), parabrachial nucleus (Krout and Loewy, 2000a),and superior colliculus (Krout et al., 2001).

MATERIALS AND METHODS

Stereotaxic injections of CTb (List Biological, Inc.,Campbell, CA, 1% solution made in distilled water) weremade with glass micropipettes in thalamic nuclei of maleSprague-Dawley rats (n 85; 200 400 g; Simonsen Lab-oratories, Gilroy, CA) by using coordinates derived fromthe rat atlas of Paxinos and Watson (1997). Iontophoreticejections were made by using 7 A on/off pulses delivered

from a Midguard precision current source (Stoelting,Wood Dale, IL) for 15 minutes. Five to 9 days later, theserats were anaesthetized with sodium pentobarbital (50mg/kg, i.p.) and pseudorabies virus was injected intothe stellate ganglion. After 5 additional days (10 14days after the CTb injections), the animals were re-anesthetized and perfused transcardially with 0.9% salinesolution followed by 500 ml of 4% paraformaldehyde solu-tion (pH 7.4). The brains were stored for at least 2 days infixative before being cut in the transverse plane at 50 mon a freezing microtome. The sections were collected in 0.1M Na

2HPO

4buffer containing 0.1% sodium azide (pH 7.4).

As determined by immunohistochemical methods (seeKrout and Loewy, 2000b, for details), some of the animals(30%) showed no signs of viral infection. The pattern of

CTb-positive cells in animals with viral infections wasvirtually identical to those animals that had no infection.The results of the viral experiments will be reported in aseparate communication.

The 85 cases used in the present account were selectedfrom material used in previous reports (Krout and Loewy,2000a,b; Krout et al., 2001) on the basis that their injec-tion sites were restricted mainly to one thalamic nucleus.Injection sites were drawn by using a camera lucida,scanned into a computer, and traced in CorelDraw8 (CorelCorp., Ottawa, Ontario). The targets included the centrallateral (n 4), central medial (n 7), lateral parafascicu-lar (n 4), medial parafascicular (n 4), oval paracentral(n 4), paracentral (n 6), anteroventral (n 3), inter-mediodorsal (n 5), mediodorsal (n 3), paratenial (n

5), rhomboid (n 4), reuniens (n 6), submedius (n 5),caudal ventromedial (n 4), and parvicellular part of theventral posteromedial (n 4) thalamic nuclei, as well asthree levels of the paraventricular thalamic nucleus: an-terior (n 6), middle (n 5), and posterior (n 6). Thethalamic nuclei were delineated according to anatomicdescriptions published by Berendse and Groenewegen(1990) and Paxinos and Watson (1997). Several examplesof injection sites are shown in Figure 1.

A one-in-fi ve series of transverse sections through theentire brainstem from these cases was processed immu-nohistochemically by the avidin-biotin complex (ABC)method for visualization of CTb retrogradely labeled neu-rons. First, sections were placed in a 1:40,000 solution of

goat anti-CTb solution (List) made in 0.1 M Na2HPO4buffer containing 0.1% sodium azide and 0.3% Triton

X-100 (Sigma, St. Louis, MO) (pH 7.4) for 2 days. Sectionswere washed in KPBS (0.02 M potassium phosphate buffer;pH 7.4), incubated in biotinylated donkey anti-goat antibod-ies (1:500; Jackson ImmunoResearch Laboratories, WestGrove, PA) for 3 hours, washed, and moved into ABC solu-tion (Vector Laboratories, Burlingame, CA) for 1 hour. CTb-labeled neurons were visualized with diaminobenzidine(DAB; Sigma Fast; Sigma; 1 tablet/15 ml of distilled water),mounted on gelatin-coated slides, and dried. The tissueswere gold-intensified (modified from Kitt et al., 1994; seeKrout et al., 1998, for details), counterstained with 0.6%thionin (Fischer, Fair Lawn, NJ), and cover-slipped withDPX mounting medium (BDH Limited, Poole, UK). The dis-tribution of the retrogradely labeled cells within the brain-stem was mapped by using an X-Y plotting system (MDplot

ver. 3.3, AccuStage, St. Paul, MN).The purpose of this study was to provide a qualitative

map of the inputs from the entire brainstem to the midlineand intralaminar thalamus. Thus, the semiquantitative

data in Table 1 are designed to highlight the patterninnervation and not present absolute numbers of neurons.Even for cases with the same injection target, small vari-ations in injection site placement, injection volume, anduptake and transport of the CTb result in a slight varia-tion in the number of retrogradely labeled cells. In addi-tion, although it did not appear to be a problem in thepresent study, CTb can be taken up by fibers of passage(for further discussion, see Luppi et al., 1990; Chen and

Aston-Jones, 1995; Ruigrok et al., 1995; Krout and Loewy,2000a,b; Krout et al., 2001). As an approximation, thenumber of labeled neurons in each brainstem nucleus wastranslated into a five point scale: 01 cells none (), 25cells light (), 6 15 cells moderate (), 16 25cells dense (), 26 or more cells very dense

(). The average density of CTb labeling from allcases with a particular injection target is given in Table 1.The pattern of brainstem3thalamic innervation is fur-ther illustrated in retrograde cell body labeling maps fromrepresentative cases that were transposed onto standard-ized drawings of the brainstem (modified from Paxinosand Watson, 1997) with the aid of a camera lucida (Figs.4 20).

The research described in this report was reviewed andapproved by the Washington University School of Medi-cine Animal Care and Biological Safety Committees andconformed to NIH guidelines.

RESULTS

Because the results of the present experiments areextensively detailed in Table 1 and in Figures 220, thissection will present only an overview of the results.However, these figures present the data throughout theentire brainstem from a representative case of eachthalamic injection target. In addition, several examplesof retrograde cell body labeling are presented in Figures2 and 3. Finally, the thalamic afferents from otherbrainstem sites, viz., periaqueductal gray matter, para-brachial nucleus, and superior colliculus, have beendescribed previously (Krout and Loewy, 2000a,b; Kroutet al., 2001).

CTb injections were made in 18 different thalamic sites.Each injection site was composed of two areas: the core

55BRAINSTEM-THALAMIC CONNECTIONS

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TABLE1.

Summaryo

fRetrogra

de

Ce

llBo

dy

La

be

ling

inthe

Bra

inste

m1

Tha

lam

icNuc

lei

Intra

lam

inar

Midline

Other

CI

CM

IPF

mPF

OPC

PC-c

PC-

r

PVA

PVT

PVP

IMD

MD

PT

Rh

Re

SM

AV

VPoc

VMc

Reticu

lar

formation

Me

du

llary

MDd

MDv

SRD

Gi

GiA

DPGi

LPGi

Pontine

IRt

PCRt

Pn

C

Pn

V

Dm

Tg

Pn

O

Su

bC

Mescencep

ha

lic

SPTg

Dp

Me

RRF

VTA

PPTg

CTF

Rap

hesystem

RMg

ROb

RPa

ll

RIP

DRc

DRd

DRv

DRv

l

MR

CLiR

RLiR

Pm

R

Othernuc

lei

Me

du

llary

Sp5C

Sp5I

RVLM

Cu

Gr

NTS

Ve

Pr

Sp5O

Pontine

A5

NI

Pr5

LC

Bar

LDTg

Mesencep

ha

lic

Cn

F

DTg

PL

IP

PBP

SNpr

SNpc

Dk

INC

1Datarepresentedonafive-po

intsca

leforthe

num

bero

flabe

ledneurons:,

none;,

lig

ht;,m

oderate;,

dense;,

morethan26ce

lls.F

ora

bbrev

iations,

see

list.


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region where neurons were filled with tracer, and a haloregion where CTb was found only in the neuropil. Theinjection targets were divided into the intralaminar tha-lamic nuclei central lateral (CL, Fig. 4), central medial(CM, Fig. 5), lateral parafascicular (lPF, Fig. 6), medialparafascicular (mPF, Fig. 7), oval paracentral (OPC, Fig.8), and paracentral nuclei (PC, Fig. 9), the midline tha-lamic nucleianterior paraventricular (PVA, Fig. 10),middle paraventricular (PVT, Fig. 11), posterior paraven-tricular (PVP, Fig. 12), intermediodorsal (IMD, Fig. 13),mediodorsal (MD, Fig. 14), paratenial (PT, Fig. 15), reuni-

ens (Re, Fig. 16), rhomboid (Rh, Fig. 17), and submediusnuclei (SM, Fig. 18), and other thalamic nucleianteroventral (AV), parvicellular part of the ventral pos-teromedial (VPpc, Fig. 19), and caudal ventromedial nu-clei (VMc, Fig. 20). Some of these cases were used inprevious publications (Krout and Loewy, 2000a,b; Kroutet al., 2001).

Intralaminar thalamic nuclei

All intralaminar nucleus cases contained a dense to very dense concentration of CTb-positive neurons in thedeep mesencephalic reticular nucleus. Labeling in otherreticular formation sites was more varied, but, in general,fewer cells were identified after PC injections and more

after lPF and mPF injections. In the raphe system, onlymPF injections resulted in a moderate number of labeledcells in the raphe pallidus nucleus. The dorsal raphe nu-cleus was labeled in every intralaminar experiment, andthe median raphe nucleus contained a moderate numberof labeled neurons in the CL, CM, lPF, and mPF experi-ments.

Every intralaminar target resulted in retrograde la-beling in the substantia nigra and the locus coeruleusnucleus. The laterodorsal tegmental nucleus, dorsal teg-mental nucleus, and the cuneiform nucleus were most

heavily labeled after lPF, mPF, CM, and CL injections.Injections in the mPF and lPF resulted in CTb-positiveneurons in a cell-poor area sandwiched between thepars compacta of the substantia nigra and the mediallemniscus (Figs. 6B, 7B). This region has been identifiedby Paxinos et al. (1999) as the parabrachial pigmentednucleus. In addition, a few cells were scattered in thenucleus of the solitary tract (NTS), vestibular nuclei,and prepositus nucleus, particularly after lPF injec-tions.

Several differences were found in the pattern of labelingbetween PC cases that had injections in the caudal PC (atthe level where the OPC is still present; n 2) and thosewith injections in the middle or rostral part of PC (n 4).

Fig. 1. A series of photomicrographs showing the extent of the cholera toxin -subunit injection sitein the anterior paraventricular thalamic nucleus (PVA; case 4312) (A), rostral paracentral thalamicnucleus (PC; case 3907) (B), middle paraventricular thalamic nucleus (PVT; case 4318) (C), and,

intermediodorsal thalamic nucleus (IMD; case 3912) (D). For abbreviations, see list. Scale bar 1 mm.


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In addition to the dense, specific projection from the in-ternal lateral parabrachial subnucleus to the caudal por-tion of PC (see Krout and Loewy, 2000a, for discussion),caudal PC injections resulted in more retrograde labelingin several other brainstem sites (see Table 1). For exam-

ple, the dorsomedial tegmental area, pedunculopontinetegmental area, oral pontine reticular nucleus, and thepars reticulata portion of the substantia nigra each hadmore labeled cells after caudal PC injections than aftermore rostral PC injection sites.

Fig. 2. Photomicrographs of a case that had cholera toxin-subunit injected into the lateral parafascicular thalamic nucleus(case 3311). A: A section through the midbrain where the outlinedregion is enlarged in B and shows ipsilateral retrograde cell body

labeling in the deep mesencephalic nucleus. C: A section through thelower brainstem where the outlined region is enlarged in D and showsa contralateral retrograde cell body labeling in the dorsal paragigan-tocellular nucleus. Scale bars 1 mm in A,C, 100 m in B,D.


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Midline thalamic nuclei

Few medullary or pontine reticular areas were la-beled after midline thalamic injections. One exceptionwas the Rh experiments. In these cases, a large number

of labeled cells were found in the gigantocellular retic-ular, caudal pontine reticular, and dorsomedial tegmen-tal nuclei. More robust labeling was identified at thelevel of the midbrain, where the majority of midline

Fig. 3. A: Photomicrograph of a case that had cholera toxin-subunit (CTb) injected into the reuniens thalamic nucleus (case4466), illustrating retrograde cell body labeling in the contralateralpedunculopontine tegmental nucleus. B: Greater detail of the outlinedregion in A. C: Photomicrograph of a case that had CTb injected into

the parvicellular portion of the ventral posterior thalamic nucleus(case 3211) illustrating contralateral retrograde cell body labeling inthe caudal part of the spinal trigeminal nucleus. D: Greater detail ofthe outlined region in C. Scale bars 1 mm in A,C, 100 m in B,D.


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Fig. 4. A: CTb injection site in the central lateral thalamic nu-cleus. BQ: Pattern of retrograde cell body labeling from case 2227.(Note: In Figures 4-20, data are plotted on standardized transverse

sections of the brainstem that were modified from Paxinos and

Watson, 1997.) In levels B-G, one triangle represents three labeledneurons. In levels H-Q, one dot represents one labeled neuron. Ap-proximate bregma levels are given on the lower right-hand corner of

each section. For abbreviations, see list.

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Figure 4 (Continued)

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Fig. 5. A: Cholera toxin -subunit injection site in the central medial thalamic nucleus. BQ: Pattern

of retrograde cell body labeling from case 3886. For abbreviations, see list.

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Fig. 6. A: Cholera toxin -subunit injection site in the lateral parafascicular thalamic nucleus.

BQ: Pattern of retrograde cell body labeling from case 3313. For abbreviations, see list.

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Fig. 7. A: Cholera toxin -subunit injection site in the medial parafascicular thalamic nucleus.


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Fig. 8. A: Cholera toxin -subunit injection site in the oval paracentral thalamic nucleus.


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Fig. 9. A: Cholera toxin -subunit injection site in the paracentral thalamic nucleus. BQ: Pattern of

retrograde cell body labeling from case 3907. For abbreviations, see list.

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Fig. 10. A: Cholera toxin -subunit injection site in the anterior paraventricular thalamic nucleus.


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Fig. 11. A: Cholera toxin -subunit injection site in the middle paraventricular thalamic nucleus.


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Fig. 12. A: Cholera toxin -subunit injection site in the posterior paraventricular thalamic nucleus.


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Fig. 13. A: Cholera toxin -subunit injection site in the intermediodorsal thalamic nucleus.


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Fig. 14. A: Cholera toxin -subunit injection site in the mediodorsal thalamic nucleus. BQ: Patternof retrograde cell body labeling from case 2516. For abbreviations, see list.

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Fig. 15. A: Cholera toxin -subunit injection site in the paratenial thalamic nucleus. BQ: Pattern of


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Fig. 16. A: Cholera toxin -subunit injection site in the reuniens thalamic nucleus. BQ: Pattern ofretrograde cell body labeling from case 3005. For abbreviations, see list.

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Fig. 17. A: Cholera toxin -subunit injection site in the rhomboid thalamic nucleus. BQ: Pattern of


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Fig. 18. A: Cholera toxin -subunit injection site in the submedius thalamic nucleus. BQ: Pattern of


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Fig. 19. A:Cholera toxin -subunit injection site in the parvicellular part of the ventral posteromedial

thalamic nucleus. BQ: Pattern of retrograde cell body labeling from case 3211. For abbreviations, seelist.

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Fig. 20. A: Cholera toxin -subunit injection site in the caudal ventromedial thalamic nucleus. BQ:

Pattern of retrograde cell body labeling from case 2930. For abbreviations, see list.

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thalamic targets resulted in CTb transport to the deepmesencephalic reticular and pedunculopontine tegmen-tal nuclei.

The pattern of labeling in the raphe system was largelysimilar to that of the intralaminar thalamic nuclei,namely, numerous cells in the dorsal raphe nucleus. Inparticular, the almost exclusive brainstem projection tothe PT was from the caudal and dorsal portions of thedorsal raphe nucleus (in addition to a moderate concen-tration of cells in its ventral and ventrolateral portions)(Fig. 15). Raphe pallidus neurons were labeled after injec-tions in the PVT system. The median raphe nucleus waslabeled after CTb injections in every midline thalamic site.In addition, the MD experiments resulted in a dense con-centration of labeled neurons in the caudal and rostrallinear raphe nuclei.

A few CTb-positive neurons in the spinal trigeminalnucleus were observed in the Re and SM cases. NTSlabeling was found predominately in the medial and com-missural NTS subnuclei after CTb injections in all por-tions of the PVT as well as after Rh and Re injections.

Every midline site resulted in labeling in the locus coer-uleus and laterodorsal tegmental nuclei; and, all midlinethalamic sites (except for the PT) received a projectionfrom the cuneiform nucleus. The parabrachial pigmentednucleus was densely labeled after PVP (Fig. 12B) and Re(Fig. 16B) injections and moderately labeled in the MD(Fig. 14B) cases. The only dense substantia nigra labelingwas found in its pars reticulata after Rh injections.

Other thalamic nuclei

A simple progression in the overall number of CTb-positive cells was observed among these three nuclei: VMchad the most labeled cells, followed by VPpc, with very fewbrainstem neurons projecting to the AV. Most reticularformation areas had at least moderate labeling after VMc

injections (Fig. 20), but only the parvicellular reticularnucleus, deep mesencephalic reticular nucleus, peduncu-lopontine tegmental nucleus, and central tegmental fieldwere at least moderately labeled in the VPpc cases (Fig.19).

Each of the VPpc cases resulted in a dense concentra-tion of labeled neurons along the dorsolateral limit of thecaudal part of the spinal trigeminal nucleus and, morerostrally, in the principal sensory trigeminal nucleus (Fig.19 J,K,P,Q). A few labeled neurons were found in thecaudal spinal and principal sensory portions of the trigem-inal nucleus in the VMc experiments. These same twotargets (VMc and VPpc) also resulted in cuneiform nu-cleus, laterodorsal tegmental nucleus, locus coeruleus, A5region, NTS, and cuneate nucleus labeling. Finally, the

pars reticulata portion of the substantia nigra was foundto contain a very dense concentration of CTb-positive neu-rons after VMc injections (Fig. 20BD).

DISCUSSION

The present results demonstrate that each of the mid-line and intralaminar thalamic nuclei receives input froma selective set of brainstem nuclei. This study took advan-tage of a collection of over 400 rat brains in which CTb wasiontophoresed into the thalamus. From this library, wechose 85 key cases with injections centered in a singlemidline or intralaminar thalamic nucleus that had mini-mal involvement of adjacent cell groups (see Krout and

Loewy, 2000a,b; Krout et al., 2001). These allowed us toobtain sample sizes of three to seven cases for each tha-lamic target to make a detailed analysis of the distributionof the brainstem cells that project to each midline andintralaminar thalamic nucleus.

The discussion will be organized in two parts: (1) acomparison of the present data with previous anterogradeand retrograde tracing investigations; and (2) a functionalinterpretation of these brainstem inputs based on thecerebral cortical targets of the thalamic nuclei (Berendseand Groenewegen, 1991; Groenewegen and Berendse,1994; Moga et al., 1995).

Comparison to previous studies

Reticular formation. Few studies have examined theascending projections of individual nuclei of the reticularformation in the rat. Most anterograde tracer studies wereinconclusive, because the injections used in these investi-gations involved multiple reticular formation nuclei (e.g.,Zemlan et al., 1984; Jones and Yang, 1985; Vertes et al.,1986). Although they confirm that there is a significant

projection from the brainstem reticular formation to themidline and intralaminar thalamic nuclei, few point-to-point details can be obtained from these reports. For ex-ample, anterograde injections involving the dorsal gigan-tocellular, gigantocellular, and the alpha part of thegigantocellular reticular nuclei indicated that these areasproject to almost all midline and intralaminar thalamicsites except the PVT, SM, MD, and PT (Jones and Yang,1985; also Vertes et al., 1986). These findings were con-firmed by the present retrograde results. However, thesize of injections used in these anterograde studies do notallow for the analysis of more subtle distinctions, such asthe very dense projection from the dorsal gigantocellularreticular nucleus to the lPF (Fig. 6) or that Rh receivesinputs predominantly from the gigantocellular reticular

nucleus but not surrounding reticular regions (Fig. 17).One of the exceptions to this trend of large reticularformation injection sites is a study by Villanueva et al.(1998) where small injections of Phaseolus vulgarisleucoagglutin in (PHA-L) were made in the subnucleusreticularis dorsalis (SRD) and cuneate nucleus (also Ber-nard et al., 1990). The complete retrograde mapping of theinputs to individual midline and intralaminar thalamicnuclei provided in the present report was extremely con-sistent with this anterograde tracer study. Dense concen-trations of labeled cells were found in the SRD and cu-neate nucleus after lPF and VMc injections, whereasmoderate to light labeling was observed in the mPF, OPC,PC, Re, Rh, and VPpc cases.

Another study made restricted injections of [3H]leucine

in the mesencephalic or pontine reticular formation andexamined the ascending projections (Vertes and Martin,1988). The results of injections in the caudal pontine andoral pontine reticular formation were very similar to eachother and matched almost exactly the pattern of innerva-tion demonstrated by the CTb method used here. Thesesimilarities include not only a dense projection from bothreticular formation areas to the lPF and mPF (amongother sites), but also parallel our finding that PVA andPVP, but not PVT, receive a slight projection from thecaudal pontine reticular nucleus.

The deep mesencephalic reticular nucleus projectsheavily to all intralaminar thalamic nuclei, including sitesthat were not heavily innervated by the caudal or oral


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pontine reticular nuclei, such as the OPC and PC. Inaddition, most midline thalamic sites, including the PVTsystem, IMD, Rh, and Re are also densely innervated.These results correlate well with anterograde tracing ex-periments (Vertes and Martin, 1988) with a single excep-tion: our data demonstrate a moderate projection from thedeep mesencephalic reticular nucleus to MD, whereas

Vertes and Martin (1988) showed no innervation of thiscell group. This discrepancy is likely due to (1) slightspread of our CTb injection sites into either the PVTsystem or CL, both of which had a dense innervation fromthe deep mesencephalic reticular nucleus; or (2) differ-ences in the demarcation of the border between CL andMD, which can be particularly difficult at rostral levels ofthe thalamus.

Substantia nigra. Substantia nigra3thalamic projections have been investigated by several groups (e.g.,Deniau and Chevalier, 1992; Nishimura et al., 1997; Sakaiet al., 1998; Groenewegen et al., 1999; Tsumori et al.,2000). The anterograde data presented in these reportssuggest a dense nigrothalamic projection to the lPF, mPF,

CL, OPC, VMc, and Rh. The retrograde data reported hereagree with these studies. In addition to these generalpatterns of innervation, the projections of subcomponentsof the substantia nigra to specific midline and intralami-nar thalamic sites have been confirmed. For example, theCL receives inputs predominately from the reticular, butnot the compact, part of substantia nigra (Fig. 1; Deniauand Chevalier, 1992). In addition, both Tsumori et al.(2000) and Deniau and Chevalier (1992) demonstrated aprojection from the caudal dorsolateral portion of the re-ticular part of substantia nigra to the OPC (althoughthese studies refer to the OPC as either the ventrolateral[Tsumori] or rostroventral [Deniau] parafascicular tha-lamic nucleus). Our data confirm these findings (see Fig.8C,D).

One prominent discrepancy can be noted in the projec-tion from the substantia nigra to the MD. Anterogradetracer studies using PHA-L demonstrated a projectionfrom the substantia nigra to the lateral and medial seg-ments of the MD (e.g., Deniau and Chevalier, 1992; Groe-newegen et al., 1999). Our data showed no retrogradelylabeled cells in substantia nigra after MD injections.These CTb injections were located in the rostral half ofMD and either excluded or only slightly involved its lat-eral segment; however, each injection encompassed themedial segment. One possible explanation for the lack oflabeling is that the nigral projection to MD innervates thecaudal portion of MD more heavily than its rostral portion(Deniau and Chevalier, 1992; Groenewegen et al., 1999)and the CTb injections in the rostral half of the MD may

have limited uptake of the tracer by nigrothalamic fibers.Nucleus Darkschewitsch. To date, only one study hasinvestigated the thalamic projections of the nucleus Dark-schewitsch with anterograde tracing methods in the rat(Krout and Loewy, 2000b). This report and the presentdata both showed that the predominate thalamic targetsare the lPF and CL. Other innervated sites included the

VMc, Re, and Rh. In addition, both reports demonstratedonly a very slight projection from the nucleus Darksche-witsch to the OPC, PC, CM, and PVP. The anterogrademap presented in the report by Krout and Loewy (2000b)correlates well with the present CTb data.

Pedunculopontine tegmental nucleus. As shownhere, the pedunculopontine tegmental nucleus innervates

all midline and intralaminar thalamic sites, except for thePT. Anterograde PHA-L tracer experiments also showedthat this nucleus has widespread projections to these tha-lamic nuclei (Hallanger et al., 1987). Although these ex-periments did not result in anterogradely labeled axonalterminals in the lPF, subsequent anterograde tracingstudies, using biotinylated dextran amine, demonstratedthat the pedunculopontine tegmental nucleus projectsdensely to the lPF (Erro et al., 1999).

Retrorubral field. The retrorubral field projects to thesame midline and intralaminar thalamic nuclei as thepedunculopontine tegmental nucleus, but the projection isweaker. This finding is comparable to the extensive an-terograde labeling found in the midline and intralaminarthalamic nuclei after the retrorubral field was injectedwith PHA-L (Hallanger and Wainer, 1988). Their reportsuggested that the retrorubral field projects to the PC;however, we found that CTb injections centered in thisthalamic site (n 6) did not result in any labeled neuronsin the retrorubral field. Thus, it is likely that their PHA-Linjections into the retrorubral field spread into adjacent

structures such as the deep mesencephalic reticular nu-cleus or the rostral end of the pedunculopontine tegmentalnucleus, because both regions contained retrogradely la-beled cells after CTb injections in the PC.

Cuneiform nucleus. A limited number of studies haveexamined the projection from the cuneiform nucleus to thethalamus (Edwards and de Olmos, 1976; Hallanger andWainer, 1988). Our data largely agrees with these reports;namely, a moderate to dense projection to the PVT system,CL, CM, mPF, and Rh. However, a few discrepancies werenoted. First, Hallanger and Wainer (1988) showed thatthe Re and lPF were devoid of any labeled fibers (their Fig.5D), but CTb injections in these two thalamic targetsresulted in moderate retrograde cell body labeling in thecuneiform nucleus. The reason for this difference is un-

clear. Also, Hallanger and Wainer (1988) showed manylabeled terminals in the PC (their Fig. 5D,E); but, in thesix PC cases examined here, only one case had any CTb-positive cells in the cuneiform nucleus. One explanationfor this discrepancy may be in the placement of the an-terograde injection site and the definition of the cuneiformnucleus used here. As can be seen in Figure 9G, after aCTb injection in the PC, a few CTb-labeled neurons arefound just ventral and lateral to the cuneiform nucleus.This region appears to be included in the PHA-L injectionsite shown by Hallanger and Wainer (1988, their Fig. 5I).Thus the anterogradely labeled axonal terminals in thePC may originate from neurons that lie just beyond theborders of the cuneiform nucleus.

Raphe nuclei. The thalamic targets of the dorsal ra-

phe and median raphe nuclei have been extensivelymapped by using small injections of PHA-L (Vertes, 1991; Vertes et al., 1999; Krout and Loewy, 2000b). Similar tothe present retrograde tracer data, Vertes (1991) used ananterograde tracer and showed that virtually all midlineand intralaminar nuclei receive a projection from the dor-sal raphe nucleus. In addition, both studies showed thatthe PC and OPC receive input from the rostral, but not thecaudal portions of the dorsal raphe nucleus. The retro-grade tracer data reported here demonstrated that mostthalamic sites also receive inputs from the median raphe,and these data largely confirm earlier anterograde work(Vertes et al., 1999). One small point of disagreement,however, was the projection to the PC: our data point to a


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weak projection to this thalamic site, whereas Vertes et al.(1999) indicated a robust projection arising from the me-dian raphe nucleus. The source of this discrepancy isunclear.

Anterograde tracer studies have suggested that neuronsin more caudal raphe nuclei, such as the raphe magnusnucleus project to the PVT (e.g., Peschanski and Besson,1984b; Hermann et al., 1996). Our findings confirm theseresults, but also indicate that a moderate projection arisesfrom the raphe pallidus nucleus.

Laterodorsal tegmental nucleus. The laterodorsaltegmental nucleus projects to every midline/intralaminarthalamic site except for the OPC (see Fig. 10 of Krout andLoewy, 2000a, for a photomicrograph of the OPC). Thiswidespread projection correlates well with the extensivethalamic terminal labeling seen after a PHA-L injectioninto laterodorsal tegmental nucleus (Satoh and Fibiger,1986). However, this anterograde tracer study alsoshowed few labeled terminals in the PVT system, partic-ularly in the PVA, whereas CTb injections at every ros-trocaudal site throughout the PVT system consistently

resulted in dense retrograde labeling in the laterodorsaltegmental nucleus. Because a few terminals do exist in thePVT, this finding may be indicative of the sensitivity of theCTb method. Alternatively, it may indicate that a largenumber of laterodorsal tegmental neurons have a limitednumber of terminals in the PVT.

A5 and C1-3 catecholamine cell groups. Several re-ports have suggested that the PVT receives adrenergic andnoradrenergic inputs from C1-3 regions and the A5 region,respectively (Byrum and Guyenet, 1987; Phillipson andBohn, 1994). Although we did not examine the neurochem-ical properties of neurons in these areas, we were unable toidentify retrogradely labeled cells in the vicinity of either theC1-3 or A5 cell groups. The explanation for these discrepan-cies almost certainly lies in technical differences between

these reports and the present data. For the C1-3 neurons,Phillipson and Bohn (1994) used several techniques that, ascompared with the current study, significantly altered boththe number of neurons labeled and their identification asadrenergic: (1) different tracers (Fluoro-Gold or fluorescentmicrospheres vs. CTb); (2) multiple injections in the PVT(5 6 per animal vs. 1 per animal); and (3) double-labelingtechniques with antibodies against phenylethanolamineN-methyltransferase (PNMT). For the A5 region, the antero-grade tracing data from Byrum and Guyenet (1987) demon-strated a projection to the posterior but not middle or ante-rior regions of the PVT (i.e., PVP, but not PVA or PVT). Inaddition, their experiments using the retrograde transport oflatex microspheres indicated only a small projection from the

A5 region to the PVT. This finding suggests that the input

from the A5 region to the PVT system may either be verysmall or originate predominately from non-noradrenergicneurons.

Vestibular nuclei. As shown by anterograde tracingstudies, the major thalamic target of the vestibular nucleiis the lPF (Shiroyama et al., 1995, 1999; Lai et al., 2000).In addition, these reports demonstrate a vestibular termi-nation in the OPC, MD, Rh, mPF, CL, and VMc. Ourresults confirmed that the lPF receives the densest inputfrom the vestibular nuclei of any thalamic site studied.However, CTb injections in the MD and OPC did notresult in consistent labeling in the vestibular nuclei. Thelack of labeling in the present MD experiments was likelythe result of the rostrally positioned injection sites, be-

cause anterior MD areas contained few anterogradely la-beled axonal terminations (see above) (Shiroyama et al.,1999; Lai et al., 2000). The reason for the lack of CTb-positive neurons after OPC injections is not clear. Other,less robust projections from the vestibular nuclei, such asthose to the Rh, mPF, CL, and VMc, were each confirmedby our retrograde tracing data.

Nucleus of the solitary tract. Anterograde axonaltracing methods have been used to show that the mainmidline/intralaminar target of the NTS is the PVT system,including its posterior, middle, and anterior subdivisions(Ricardo and Koh, 1978; Ter Horst and Streefland, 1994;Ruggiero et al., 1998). These reports have also indicatedthat a few scattered terminal fibers were present in theRe, CM, Rh, lPF, mPF, VMc, and OPC. Our data agreewith these findings both in terms of location and intensityof innervation.

However, at least one difference was noted. In the VPpcexperiments, NTS neurons were labeled. Earlier antero-grade tracing studies failed to find terminal axonal label-ing in this region (Ricardo and Koh, 1978; Ter Horst and

Streefland, 1994; Ruggiero et al., 1998). However, Ruggi-ero et al. (1998) found dense terminal labeling ventral tothe fasciculus retroflexus and identified this region as the

ventral sector of the parafascicular nucleus. This areamay correspond to the region we have defined here as the

VPpc.

Functional considerations

For much of past half-century, the midline and in-tralaminar thalamic nuclei were thought to be a nonspe-cific relay center that globally activated the cerebral cor-tex in response to signals from the brainstem reticularformation (e.g., Lorente de No, 1938; Moruzzi and Ma-goun, 1949; Jones and Leavitt, 1974). This concept haschanged. Neuroanatomic studies have now shown that the

efferent projections from these thalamic nuclei to the ce-rebral cortex, striatum, and amygdala are highly specific(Berendse and Groenewegen, 1990, 1991; Turner andHerkenham, 1991; Groenewegen and Berendse, 1994;Moga et al., 1995). More recent hypotheses propose a rolefor brainstem neurons not only in arousal and sleep/wakefulness cycles, but also in attention, homeostasis,and emotion (e.g., Berendse and Groenewegen, 1990,1991; Dostrovsky and Guilbaud, 1990; Powell et al., 1990;Grunwerg and Krauthamer, 1992; Groenewegen and Be-rendse, 1994; Gottesmann, 1999).

Because more than 50 brainstem nuclei provide inputsto the midline and intralaminar thalamic nuclei, a com-prehensive discussion of each of these sites is beyond thescope of this study. However, the present report, along

with the others in this series (Krout and Loewy, 2000a,b;Krout et al., 2001), support the idea that brainstem nucleicontribute to specialized forebrain pathways by means ofspecific projections to the midline and intralaminar tha-lamic nuclei. To focus attention on several major issues,this discussion will concentrate on arousal, motor, somato-sensory, and visceral systems.

Although functional interpretations can be deducedfrom the anatomic connections, there is a paucity of phys-iological observations regarding brainstem3thalamic3forebrain circuits. In addition, because the concept of thenonspecific arousal system had dominated the thinking inthis field, the midline and intralaminar thalamic nucleiwere often considered as a single functional unit with the


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same function and connectivity. This intellectual bias hasinfluenced physiological experiments that were designedto dissect the role(s) of individual thalamic nuclei inarousal, cognition, and other higher-level brain activities,such as memory (but see Mair, 1994; Van Der Werf et al.,1999, 2000; Jeljeli et al., 2000).

Arousal systems. Several lines of evidence have im-plicated the medial thalamus and reticular formation inmodulating arousal. Electrical stimulation of the in-tralaminar thalamus in anaesthetized animals producesEEG desynchronization (Morison and Dempsey, 1942;Hunter and Jasper, 1949), an effect duplicated by electri-cal stimulation of the reticular formation (Moruzzi andMagoun, 1949). This effect has been shown more directlyby studies which show that the reticular formation caninfluence the cerebral cortex and other forebrain regionsthrough connections with the intralaminar thalamus (Ste-riade, 1996, 1999; Erro et al., 1999). In humans, PETstudies demonstrated that activation of both the reticularformation and intralaminar nuclei occurs during tasksrequiring arousal/attention (Kinomura et al., 1996). In

addition, thalamic relay cells change their activity, de-pending on whether the animal is awake or asleep (Glennand Steriade, 1982; Steriade and Glenn, 1982; Steriade,1999). Unfortunately, most of these studies have relied ontechniques that stimulate fibers of passage or that simplycorrelate brainstem and cortical activity without showinga causal relationship.

Because the present study has shown that midline andintralaminar thalamic nuclei receive robust inputs fromnonreticular areas, other brainstem cell groups, besidesthe reticular formation, may also be important in thalamiccontrol of arousal. Three examples can be cited. First, thelocus coeruleus provides a noradrenergic input to thala-mus and cerebral cortex (Jones and Moore, 1977; Loughlinet al., 1982; Peschanski and Besson, 1984a), and the dis-

charge rate of these neurons increases in response to novelstimuli and decreases during sleep (Aston-Jones andBloom, 1981; Foote et al., 1983). Because lesions of thisregion do not affect the sleep-wake cycle (Jones andMoore, 1977), the locus coeruleus has been proposed toplay a role in the maintenance, but not initiation, ofarousal. Second, dorsal raphe neurons display similarproperties to those seen in the locus coeruleus: they de-crease their firing rate during slow wave sleep. However,lesions of the dorsal raphe nucleus produce a differenteffect than locus coeruleus lesions, viz., they cause insom-nia (Lydic et al., 1987; for review see Robbins et al., 1998).Third, other cell groups important in maintaining normaltransitions in the level of arousal include the laterodorsaltegmental nucleus and the pedunculopontine nucleus that

provide cholinergic inputs to the thalamus and brainstemand have been implicated in the switch from slow-wave torapid eye movement sleep (Edley and Graybiel, 1983; Ryeet al., 1987, 1988; Steriade et al., 1990; Bolton et al., 1993).

In the present study, we have demonstrated that thedeep mesencephalic reticular, laterodorsal tegmental, pe-dunculopontine tegmental, dorsal raphe, and locus coer-uleus nuclei project to every midline and intralaminarthalamic site (Fig. 21). Overlapping distributions of la-beled neurons in the deep mesencephalic reticular nucleuswere observed after the various thalamic injections. How-ever, the present experimental design did not examinemultiple thalamic sites with double-tracer techniques;thus, it is not known whether subsets of thalamic nuclei

receive inputs from single deep mesencephalic reticularnucleus neurons or whether there is a segregation of thal-amically projecting neurons in this region. This issue isimportant because, if it is shown that single brainstemneurons innervate multiple thalamic sites, then it could bepredicted that these cells would influence multiple areasof the cerebral cortex. This possibility may be particularlyrelevant, because the deep mesencephalic reticular andpedunculopontine tegmental nuclei are the brainstem re-gions that were stimulated by Moruzzi and Magoun (1949)to produce EEG desynchronization.

Changes in arousal modify the responsiveness of tha-lamic neurons to stimuli (Glenn and Steriade, 1982; Ste-riade and Glenn, 1982). For example, stimulation in thearea of the cholinergic neurons of the pedunculopontinetegmental and laterodorsal tegmental nuclei during sleeppotentiated the response of thalamic neurons to somaticstimulation (Pare and Steriade, 1990; Steriade, 1996).

Also, ascending inputs to the intralaminar nuclei mayplay a role in modulating information flow in the cerebralcortex, because stimulation of the mesencephalic reticular

formation facilitated oscillation between two visual areas(Munk et al., 1996). Thus, the level of brainstem activa-tion may affect thalamocortical, thalamostriatal, or evencorticocortical transmission.

Motor systems. Several midline and intralaminarthalamic sites, particularly the lPF and CL, may play akey role in movement. Their contribution to motor activitycan be deduced from their anatomic connectivity as well asby lesion and stimulation studies. The lPF projects notonly to motor-related areas of cerebral cortex and striatumbut also to the subthalamic nucleus (Berendse and Groe-newegen, 1990, 1991; Mouroux and Feger, 1993). Becausestimulation of the lPF inhibited movement in free-roaming rats (Mileikovsky et al., 1994), we have previ-ously suggested that this lPF3subthalamic circuit may be

related to stopping ongoing behavior and allowing theinitiation of new motor programs (Krout et al., 2001). Inaddition to these efferent projections, the lPF receivesinputs from motor related brainstem sites, including thesubstantia nigra, nucleus Darkschewitsch, superior col-liculus, cuneiform nucleus, vestibular nuclei, and dorsalcolumn nuclei (see Table 1) (Krout and Loewy, 2000b; Laiet al., 2000; Krout et al., 2001). However, the lPF alsoreceives projections from a wide range of other brainstemareas that have been associated with other modalitiessuch as autonomic function (e.g., rostral ventrolateral me-dulla and NTS), somatosensory function (e.g., spinal tri-geminal nucleus), and some sites that integrate these andother functions (e.g., periaqueductal gray matter andSRD) (Roy et al., 1992; Behbehani, 1995; Krout and

Loewy, 2000b). These inputs suggest that many differenttypes of information affect motor control at this level of theneuraxis.

The CL has also been associated with motor control. Forexample, bilateral lesions of CL result in deficits in aspecialized motor task called the rotorod test but not inthe other motor tasks (Jeljeli et al., 2000). This deficit wasinterpreted to be related to disruption of a cerebellar3CLpathway and not to a generalized decrease in attention orarousal. However, this interpretation is complicated bythe fact that CL also receives dense projections frombrainstem structures such as the superior colliculus, peri-aqueductal gray matter, cuneiform nucleus, nucleus Dark-schewitsch, and several other nuclei (see Table 1) (Yamasaki


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Fig. 21. Summary figure illustrating a subset of arousal-relatedbrainstem nuclei that innervate almost every midline and intralami-nar thalamic nucleus. Thalamic nuclei situated on the midline and

the caudal thalamic nuclei also receive visceral-related inputs. Thesedata suggest that convergent arousal/visceral information may affectlarge regions of cerebral cortex (top panel). For abbreviations, see list.


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and Krauthamer, 1990; Grunwerg and Krauthamer, 1992;Krout and Loewy, 2000b; Krout et al., 2001). It is unclearhow these multiple sites may contribute to altering motorcommand signals at both the cortical and striatal levels.

Somatosensation and nociception. Midline and in-tralaminar thalamic nuclei neurons respond to innocuoussomatic stimuli, noxious stimuli, or, in some cases, both(e.g., Reyes-Vazquez et al., 1989; Dostrovsky and Guil-baud, 1990). These medial thalamic neurons have recep-tive fields that encompass large areas of the body, whereasprincipal thalamic relay neurons respond to stimuli ap-plied to small regions of the contralateral body (e.g., Wil-lis, 1985; Monconduit et al., 1999). This finding suggests afundamental difference in the functions of these two path-ways.

Somatic information reaches the thalamus by means ofseveral neural circuits. Anatomic studies have shown adirect projection from the dorsal horn of the spinal cord toall intralaminar and most midline thalamic sites and thisascending system likely transmits somatosensory and vis-ceral information (Cliffer et al., 1991). Here, we have

demonstrated that only a few thalamic regions may re-ceive somatic inputs from the spinal trigeminal nucleus;these include the VPpc, VMc, lPF, mPF, and OPC. Otherstudies have suggested that somatosensory/nociceptive in-formation may also reach the thalamus by indirect means,for example, by means of relays in the parabrachial nu-cleus (Bester et al., 1999; Krout and Loewy, 2000a) orbrainstem reticular nuclei (Peschanski and Besson, 1984c;Bernard et al., 1990; Roy et al., 1992; Villanueva et al.,1998). Whatever the circuits involved, the large receptivefield sizes found in midline/intralaminar thalamic neu-rons suggest that this system is not required for localizingstimuli but may be responsible for modulating orienting orarousal responses (Orem et al., 1973; Maldonado andSchlag, 1984; Schlag-Rey and Schlag, 1984; Merker and

Schlag, 1985; Grunwerg and Krauthamer, 1992).Visceral systems. The present study has demon-strated that the NTS is linked to the CM, lPF, mPF, OPC,PVT system, Rh, Re, VPpc, and VMc. In addition, visceral-related parabrachial subnuclei also project to these samesites (see Krout and Loewy, 2000a). Based on the efferentterminations of these thalamic nuclei, these data suggestthat visceral information could reach medial prefrontal,anterior cingulate, motor, perirhinal, and insular cortices(see Fig. 21), as well as other forebrain structures such asthe amygdala, ventral subiculum, and striatum (Herken-ham, 1979; Arbuthnott et al., 1990; Berendse and Groe-newegen, 1990, 1991; Groenewegen et al., 1990; Turnerand Herkenham, 1991; Moga et al., 1995). These findingssuggest that the hypothesis proposed by Neafsey (1990)

that the primary viscerosensory area is only the insularcortex (see also Cechetto and Saper, 1987; Allen et al.,1991) should be broadened, because visceral sensory in-formation may be distributed to many more cortical sitesthan had been previously postulated.

CONCLUSION

Many of the studies on the physiological properties ofthe midline and intralaminar properties have concen-trated on only one or a few modalities, such as nociception.However, the large number of brainstem nuclei labeledafter CTb injections into each thalamic site suggest thatmany different types of sensory or feedback information

impinge on a single thalamic nucleus and, perhaps, onsingle neurons. This concept is supported by anatomic andphysiological data that show that the thalamic nuclei re-ceive convergent inputs (e.g., Reyes-Vazquez et al., 1989;Grunwerg and Krauthamer, 1990, 1992; Groenewegen etal., 1999). In addition, many of the raphe system or retic-ular formation neurons that project to the thalamus arelikely to be polysensory (see Mason, 2001). Thus the func-tion of the midline and intralaminar thalamic nuclei islikely to be best understood holistically, as part of anintegrative system modulating subtle changes in behavioror attention in response to a wide range of somatosensoryand visceral signals.

ACKNOWLEDGMENTS

We thank Alex Field and Xay Van Nguyen for theirexcellent technical assistance.

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