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Efferent Projections of Reuniens and Rhomboid Nuclei of the Thalamus in the Rat ROBERT P. VERTES, 1 * WALTER B. HOOVER, 1 ANGELA CRISTINA DO VALLE, 1,2 ALEXANDRA SHERMAN, 1 AND J.J. RODRIGUEZ 1,3 1 Center for Complex Systems and Brain Sciences, Florida Atlantic University, Boca Raton, Florida 33431 2 Neuroscience Laboratory, University of Sao Paulo School of Medicine, Sao Paulo, SP 05508-900 Brazil 3 Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom ABSTRACT The nucleus reuniens (RE) is the largest of the midline nuclei of the thalamus and exerts strong excitatory actions on the hippocampus and medial prefrontal cortex. Although RE projections to the hippocampus have been well documented, no study using modern tracers has examined the totality of RE projections. With the anterograde anatomical tracer Phaseo- lus vulgaris leuccoagglutinin, we examined the efferent projections of RE as well as those of the rhomboid nucleus (RH) located dorsal to RE. Control injections were made in the central medial nucleus (CEM) of the thalamus. We showed that the output of RE is almost entirely directed to the hippocampus and “limbic” cortical structures. Specifically, RE projects strongly to the medial frontal polar, anterior piriform, medial and ventral orbital, anterior cingulate, prelimbic, infralimbic, insular, perirhinal, and entorhinal cortices as well as to CA1, dorsal and ventral subiculum, and parasubiculum of the hippocampus. RH distributes more widely than RE, that is, to several RE targets but also significantly to regions of motor, somatosensory, posterior parietal, retrosplenial, temporal, and occipital cortices; to nucleus accumbens; and to the basolateral nucleus of amygdala. The ventral midline thalamus is positioned to exert significant control over fairly widespread regions of the cortex (limbic, sensory, motor), hippocampus, dorsal and ventral striatum, and basal nuclei of the amygdala, possibly to coordinate limbic and sensorimotor functions. We suggest that RE/RH may represent an important conduit in the exchange of information between subcortical-cortical and cortical-cortical limbic structures potentially involved in the selection of appropriate responses to specific and changing sets of environmental conditions. J. Comp. Neurol. 499: 768 –796, 2006. © 2006 Wiley-Liss, Inc. Indexing terms: medial prefrontal cortex; nucleus accumbens; hippocampus; basolateral nucleus of amygdala; central medial nucleus of thalamus The nucleus reuniens (RE) lies ventrally on the midline, directly above the third ventricle, and extends longitudi- nally virtually throughout the thalamus (Swanson, 1998; Bokor et al., 2002). RE is the largest of the midline nuclei of the thalamus (Groenewegen and Witter, 2004). The rhomboid nucleus (RH) lies dorsal to RE and overlaps with approximately the caudal two-thirds of RE. The caudal part of RH has a characteristic rhomboid-like appearance (Swanson, 1998), hence its name. Based on the early demonstration that low-frequency stimulation of the midline and intralaminar nuclei of the thalamus produced slow synchronous activity over wide- spread regions of the cortex (recruiting responses; Dempsey and Morrison, 1942), the midline thalamus was viewed as “nonspecific” thalamus, exerting nonspecific or global effects on the cortical mantle (for review see Bentivoglio et al., 1991; Grant sponsor: National Institute of Mental Health; Grant number: MH63519; Grant number: MH01476. *Correspondence to: Dr. Robert P. Vertes, Center for Complex Systems and Brain Sciences, Florida Atlantic University, Boca Raton, FL 33431. E-mail: [email protected] Received 28 December 2005; Revised 25 May 2006; Accepted 29 June 2006 DOI 10.1002/cne.21135 Published online in Wiley InterScience (www.interscience.wiley.com). THE JOURNAL OF COMPARATIVE NEUROLOGY 499:768 –796 (2006) © 2006 WILEY-LISS, INC.

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Page 1: Efferent Projections of Reuniens and Rhomboid Nuclei of ...vertes/re-rh.pdf · Efferent Projections of Reuniens and Rhomboid Nuclei of the Thalamus in the Rat ROBERT P. VERTES,1*

Efferent Projections of Reuniens andRhomboid Nuclei of the Thalamus

in the Rat

ROBERT P. VERTES,1* WALTER B. HOOVER,1 ANGELA CRISTINA DO VALLE,1,2

ALEXANDRA SHERMAN,1AND J.J. RODRIGUEZ1,3

1Center for Complex Systems and Brain Sciences, Florida Atlantic University,Boca Raton, Florida 33431

2Neuroscience Laboratory, University of Sao Paulo School of Medicine, Sao Paulo,SP 05508-900 Brazil

3Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom

ABSTRACTThe nucleus reuniens (RE) is the largest of the midline nuclei of the thalamus and exerts

strong excitatory actions on the hippocampus and medial prefrontal cortex. Although REprojections to the hippocampus have been well documented, no study using modern tracershas examined the totality of RE projections. With the anterograde anatomical tracer Phaseo-lus vulgaris leuccoagglutinin, we examined the efferent projections of RE as well as those ofthe rhomboid nucleus (RH) located dorsal to RE. Control injections were made in the centralmedial nucleus (CEM) of the thalamus. We showed that the output of RE is almost entirelydirected to the hippocampus and “limbic” cortical structures. Specifically, RE projectsstrongly to the medial frontal polar, anterior piriform, medial and ventral orbital, anteriorcingulate, prelimbic, infralimbic, insular, perirhinal, and entorhinal cortices as well as toCA1, dorsal and ventral subiculum, and parasubiculum of the hippocampus. RH distributesmore widely than RE, that is, to several RE targets but also significantly to regions of motor,somatosensory, posterior parietal, retrosplenial, temporal, and occipital cortices; to nucleusaccumbens; and to the basolateral nucleus of amygdala. The ventral midline thalamus ispositioned to exert significant control over fairly widespread regions of the cortex (limbic,sensory, motor), hippocampus, dorsal and ventral striatum, and basal nuclei of the amygdala,possibly to coordinate limbic and sensorimotor functions. We suggest that RE/RH mayrepresent an important conduit in the exchange of information between subcortical-corticaland cortical-cortical limbic structures potentially involved in the selection of appropriateresponses to specific and changing sets of environmental conditions. J. Comp. Neurol. 499:768–796, 2006. © 2006 Wiley-Liss, Inc.

Indexing terms: medial prefrontal cortex; nucleus accumbens; hippocampus; basolateral nucleus

of amygdala; central medial nucleus of thalamus

The nucleus reuniens (RE) lies ventrally on the midline,directly above the third ventricle, and extends longitudi-nally virtually throughout the thalamus (Swanson, 1998;Bokor et al., 2002). RE is the largest of the midline nucleiof the thalamus (Groenewegen and Witter, 2004). Therhomboid nucleus (RH) lies dorsal to RE and overlaps withapproximately the caudal two-thirds of RE. The caudalpart of RH has a characteristic rhomboid-like appearance(Swanson, 1998), hence its name.

Based on the early demonstration that low-frequencystimulation of the midline and intralaminar nuclei of thethalamus produced slow synchronous activity over wide-spread regions of the cortex (recruiting responses; Dempsey

and Morrison, 1942), the midline thalamus was viewed as“nonspecific” thalamus, exerting nonspecific or global effectson the cortical mantle (for review see Bentivoglio et al., 1991;

Grant sponsor: National Institute of Mental Health; Grant number:MH63519; Grant number: MH01476.

*Correspondence to: Dr. Robert P. Vertes, Center for Complex Systemsand Brain Sciences, Florida Atlantic University, Boca Raton, FL 33431.E-mail: [email protected]

Received 28 December 2005; Revised 25 May 2006; Accepted 29 June2006

DOI 10.1002/cne.21135Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 499:768–796 (2006)

© 2006 WILEY-LISS, INC.

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Groenewegen and Berendse, 1994). The notion of the mid-line thalamus as “nonspecific” has been revised, however,based in large part on recent anatomical findings showingthat as a group the midline nuclei project not widelythroughout the neocortex but, rather, selectively to specificregions of the prefrontal cortex (Berendse and Groenewegen,1991; Van der Werf et al., 2002; Vertes et al., 2003; Groe-newegen and Witter, 2004).

It is well established that nucleus reuniens (RE) distrib-utes densely to the hippocampal formation (HF; Herken-ham, 1978; Wouterlood et al., 1990; Wouterlood, 1991;Bokor et al., 2002). RE axons form asymmetric (excitatory)contacts predominantly on distal dendrites of pyramidalcells in stratum lacunosum-moleculare of CA1 and thesubiculum (Wouterlood et al., 1990). RE stimulation pro-duces strong excitatory effects at CA1 of the hippocampus(Dolleman-Van der Weel et al., 1997; Bertram and Zhang,1999).

The hippocampus distributes heavily to the medial pre-frontal cortex (mPFC; Swanson, 1981; Irle and Markow-

itsch, 1982; Cavada et al., 1983; Goldman-Rakic et al.,1984; Ferino et al., 1987; Jay et al., 1989; Jay and Witter,1991; Carr and Sesack, 1996; Ishikawa and Nakamura,2003), but there are no return projections from the mPFCto the hippocampus (Goldman-Rakic et al., 1984; Room etal., 1985; Reep et al., 1987; Sesack et al., 1989; Hurley etal., 1991; Takagishi and Chiba, 1991; Buchanan et al.,1994). The recent demonstration that mPFC strongly tar-gets RE (Vertes, 2002, 2004), coupled with direct RE to HFprojections (Herkenham, 1978; Wouterlood et al., 1990;Wouterlood, 1991; Bokor et al., 2002), suggests that RE isthe main route for the actions of the mPFC on thehippocampus/parahippocampus. This system of connec-tions (mPFC-RE-HF) thus completes an important func-tional loop between HF and mPFC.

RE receives widespread, mainly limbic, input from thebrainstem, hypothalamus, amygdala, basal forebrain, andlimbic cortex (Herkenham, 1978; Risold et al., 1997; McK-enna and Vertes, 2004). RE is pivotally positioned to relaya vast array of (limbic) information to its main targets.

Abbreviations

AC anterior cingulate cortexACC nucleus accumbensACo anterior commissureAD anterodorsal nucleus of thalamusAGl lateral agranular (frontal) cortexAGm medial agranular (frontal) cortexAH anterior nucleus of hypothalamusAI,d,p,v agranular insular cortex, dorsal, posterior, ventral divi-

sionsAM anteromedial nucleus of thalamusAON anterior olfactory nucleusAPN anterior pretectal nucleusAV anteroventral nucleus of thalamusBLA basolateral nucleus of amygdalaBLAa,p BLA anterior, posterior divisionsBMA basomedial nucleus of amygdalaBST bed nucleus of stria terminalisCA1,2,3 field CA1, CA2, CA3 of Ammon’s hornCB cinguum bundleCC corpus callosumCEA central nucleus of amygdalaCEM central medial nucleus of thalamusCLA claustrumCOA cortical nucleus of amygdalaCP caudate-putamenDBh nucleus of the diagonal band, horizontal limbDG dentate gyrus of hippocampusDMh dorsomedial nucleus of hypothalamusEC,l,m entorhinal cortex, lateral, medial divisionsECld,v ECl, dorsal, ventral partsECT ectorhinal cortexEN endopiriform nucleusFP,l,m frontal polar cortex, lateral, medial divisionsGI granular insular cortexGP globus pallidusHF hippocampal formationIAM interanteromedial nucleus of thalamusIL infralimbic cortexIMD intermediodorsal nucleus of thalamusLA lateral nucleus of amygdalaLD lateral dorsal nucleus of thalamusLG,d lateral geniculate nucleus, dorsal divisionLH lateral habenulaLHy lateral hypothalamic areaLO lateral orbital cortexLP lateral posterior nucleus of thalamusLS lateral septal nucleusLV lateral ventricleMA magnocellular preoptic nucleusMB mammillary bodiesMD mediodorsal nucleus of thalamus

MFB medial forebrain bundleMO medial orbital cortexmPFC medial prefrontal cortexMPO medial preoptic areaMRF mesencephalic reticular formationMS medial septumMT mammillothalamic tractOC occipital cortexOT olfactory tuberclePA piriform-amygdaloid transition areaPAG periaqueductal grayPARA parasubiculum of HFPC paracentral nucleus of thalamusPH posterior nucleus of hypothalamusPIR piriform cortexPL prelimbic cortexPO posterior nucleus of thalamusPOST postsubiculum of HF hippocampusPPC posterior parietal cortexPRC perirhinal cortexpRE perireuniens nucleusPRE presubiculum of HFPT paratenial nucleus of thalamusPV paraventricular nucleus of thalamusPVh paraventricular nucleus of hypothalamusRE nucleus reuniens of thalamusRF rhinal fissueRH rhomboid nucleus of thalamusRSC retrosplenial cortexRSCagl,d lateral agranular, dorsal fields of RSCRT reticular nucleus of thalamusSF septofimbrial nucleusSI substantia innominataslm stratum lacunosum-moleculare of Ammon’s hornsm stria medullarisSMT submedial nucleus of thalamusSSI primary somatosensory cortexSSII secondary somatosensory cortexSUB,d,v subiculum, dorsal, ventral partsSUM supramammillary nucleusTE temporal cortexTT,d,v tenia tecta, dorsal, ventral partsVAL ventral anterior-lateral complex of thalamusVB ventrobasal complex of thalamusVLO ventrolateral orbital cortexVM ventral medial nucleus of thalamusVMh ventromedial nucleus of hypothalamusVO ventral orbital cortexVTA ventral tegmental areaZI zona incerta3V third ventricle

The Journal of Comparative Neurology. DOI 10.1002/cne

769EFFERENTS OF REUNIENS AND RHOMBOID NUCLEI

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Although RE projections to HF have been well docu-mented (Herkenham, 1978; Wouterlood et al., 1990; Suand Bentivoglio, 1990; Wouterlood, 1991; Dolleman-Vander Weel and Witter, 1996; Bokor et al., 2002), few reportshave examined RE projections to other sites (Herkenham,1978; Risold et al., 1997) or the totality of RE projections.

The rhomboid nucleus shares properties with RE. Spe-cifically, as with RE, RH receives afferents from wide-spread regions of the brainstem and forebrain (Van derWerf et al., 2002; Vertes et al., 2004b; Owens, 2005),projects to the hippocampus (Riley and Moore, 1981; Suand Bentivoglio, 1990), and exerts excitatory actions onHF (Dolleman-Van der Weel et al., 1997; Bertram andZhang, 1999). Very little is known about the connections ofRH, either inputs or outputs. To our knowledge, only asingle early report concerning the rat (Ohtake andYamada, 1989) has examined RH projections.

Based on the pivotal positions of RE and RH in thelimbic circuitry and the general lack of information ontheir connections, we sought to examine comprehensivelythe efferent projections of the reuniens and rhomboidnuclei of the midline thalamus. We found that REdistributes densely to the orbitomedial prefrontal cortex,hippocampus/parahippocampus, and some subcorticallimbic sites. RE projections are more restricted than thoseof RH. RE projects much more strongly than RH to thehippocampus and to the entorhinal cortex, whereas RHdistributes more heavily than RE to nucleus accumbens.Based on their efferent projections, RE and RH are stra-tegically positioned to influence, and possibly coordinate,the activity of select limbic subcortical and cortical struc-tures.

MATERIALS AND METHODS

Single injections of Phaseolus vulgaris leuccoagglutinin(PHA-L) were made into RE, RH, or the central medial(CEM) nuclei of the thalamus of 42 male Sprague-Dawley(Charles River, Wilmington, MA) rats weighing 275–325g. These experiments were approved by the Florida Atlan-tic University Institutional Animal Care and Use Com-mittee and conform to all Federal regulations and Na-tional Institutes of Health guidelines for the care and useof laboratory animals.

Powdered lectin from PHA-L was reconstituted to 5% in0.05 M sodium phosphate buffer, pH 7.4. The PHA-L so-lution was iontophoretically deposited in the brains ofanesthetized rats by means of a glass micropipette with anoutside tip diameter of 40–60 �m. Positive direct current(5–10 �A) was applied through a Grass stimulator (model88) coupled with a high-voltage stimulator (FHC, Bowdo-inham, ME) at 2 seconds “on”/2 seconds “off” intervals for30–40 minutes. After a survival time of 7–10 days, ani-mals were deeply anesthetized with sodium pentobarbitaland perfused transcardially with a buffered saline wash(pH 7.4, 300 ml/animal), followed by fixative (2.5% para-formaldehyde, 0.2–0.5% glutaraldehyde in 0.1 M phos-phate buffer, pH 7.4; 300–500 ml/animal) and then by10% sucrose in the same phosphate buffer (150 ml/animal). The brains were removed and stored overnight at4°C in 30% sucrose in the same phosphate buffer. Brainswere generally cut on the following day, and 40- or 50-�mfrozen sections were collected in phosphate-buffered sa-line (PBS; 0.9% sodium chloride in 0.1 M sodium phos-phate buffer, pH 7.4). A complete series of sections was

treated with 1% sodium borohydride in 0.1 M phosphatebuffer (PB) for 30 minutes to remove excessive aldehydes.Sections were washed three times for 5 minutes each (3 �5 minutes) in PB and then incubated for 30 minutes in0.5% bovine serum albumin in 0.1 M Tris-buffered saline(TBS; pH, 7.6) at room temperature (RT) to minimizenonspecific labeling. After this, sections were incubatedovernight at RT in 0.1% bovine serum albumin in TBScontaining 0.25% Triton X-100 and biotinylated goat anti-PHA-L (Vector, Burlingame, CA) at a dilution 1:500. Sec-tions were then washed in PBS (5 � 5 minutes) and placed1) for 1 hour in 1:400 dilution of biotinylated rabbit anti-goat immunoglobulin (IgG) and 2) for 1 hour in 1:200dilution of peroxidase-avidin complex from the VectorElite kit. After 5 � 5 minute rinses, sections were incu-bated in a solution containing 0.022% of 3,3� diaminoben-zidine (DAB) in PBS for 5 minutes, followed by a second5-minute DAB (same concentration) incubation to which0.003% H2O2 had been added. Sections were then rinsedagain in PBS (3 � 1 minutes) and mounted onto chrome-alum-gelatin-coated slides. An adjacent series of sectionsfrom each rat was stained with cresyl violet for anatomicalreference. Sections were examined via light- and darkfieldoptics. Injection sites and labeled fibers were plotted onrepresentative schematic transverse sections through thebrain with sections adapted from the rat brain atlas ofSwanson (1998). Material judged particularly useful foremphasizing or clarifying points of text was illustratedwith light- and darkfield photomicrographs with a NikonDXM1200 digital camera mounted on a Nikon Eclipsemicroscope. Digital images were captured and recon-structed in ImagePro and enhanced (contrast and bright-ness) in Adobe Photoshop 9.0.

RESULTS

The patterns of distribution of labeled fibers throughoutthe brain with injections in the reuniens and rhomboidnuclei of the midline thalamus are described. Figure 1depicts sites of injections in RE (Fig. 1A,B) and RH (Fig.1C,D) for the schematically illustrated cases. The patternsof labeling obtained with the schematically depicted casesare representative of patterns seen with nonillustratedcases. RE and RH cases are compared with control caseswith injections in the central medial nucleus (CEM) of thethalamus, dorsal to RH. RE (proper) cases are also com-pared with cases with injections in the perireuniens re-gion (or the wings of RE; Risold et al., 1997, Swanson,1998). Figure 2 shows cytoarchitectural boundaries of nu-clei of the midline thalamus at four levels of the thalamus(Fig. 2A–D) together with schematic depictions of repre-sentative injection sites in RE (proper), the anterior RE(aRE), perireuniens nucleus (pRE), RH, and CEM (Fig.2E–H, Table 1).

Nucleus reuniens: case RE-20

Figure 3 schematically depicts the pattern of distribu-tion of labeled fibers following a PHA-L injection in RE(case RE-20; Figs. 1A, 2F–H). Labeled fibers coursed pri-marily ventrolaterally from RE, traversing the ventrome-dial nucleus of thalamus, the zona incerta, and the dorso-lateral hypothalamus to reach the medial forebrainbundle (MFB; Fig. 3P–S) and from there followed rostral,caudal, or lateral routes. The bulk of labeled axons as-

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770 R.P. VERTES ET AL.

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cended through the lateral hypothalamus/MFB to thebasal forebrain, where they joined the internal capsulecoursing through ventromedial regions of the striatum indiscrete fascicles to the rostral forebrain (Fig. 3G–L). Atthe anterior forebrain, they either distributed terminallyto parts of the frontal cortex or turned caudally, coursingthrough the cingulum bundle to the hippocampus orthrough outer layers of the frontal and then caudal re-

gions of cortex. A second prominent bundle exited laterallyfrom the lateral hypothalamus bound for the amygdalaand ventrolateral regions of cortex bordering the rhinalfissure. Some labeled fibers of this tract continued cau-dally to reach parts of the subiculum of hippocampus. Thesmallest of the three bundles descended through lateralhypothalamus/MFB to caudal regions of the diencephalon(mainly to the hypothalamus) and to the rostral midbrain.

Fig. 1. A,C: Low-power lightfield photomicrograph showing thesites of Phaseolus vulgaris leuccoagglutinin (PHA-L) injections in thereuniens (A; case RE-20) and rhomboid nuclei (C; case RH-7) of themidline thalamus. B,D: High-magnification lightfield photomicro-

graphs from the core of injections depicting patterns of PHA-L-filledcells in RE (B) and RH (D). For abbreviations see list. Scale bar �1,000 �m for A,C; 175 �m for B,D.

The Journal of Comparative Neurology. DOI 10.1002/cne

771EFFERENTS OF REUNIENS AND RHOMBOID NUCLEI

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Fig. 2. A–D: Series of Nissl-stained sections through the thalamusshowing cytoarchitectural boundaries of nuclei of the midline thala-mus and surrounding structures. E–H: Series of matched (to stainedsections, A–D) schematic sections through the thalamus showing thelocations of injections in the rostral (case aRE-9) and caudal nucleus

reuniens (cases RE-12, RE-20, RE-A5), perireuniens (cases pRE-1,pRE-60), rhomboid nucleus (cases RH-4, RH-7, RH-J5), and centralmedial nucleus of thalamus (case CEM-10). For abbreviations see list.Scale bar � 1,000 �m for A–D.

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Labeling within anterior levels of the forebrain (pre-

septum; Fig. 3A–H). As illustrated in Figure 3A–H, la-beling was prominent within the rostral forebrain. At theanterior pole of the forebrain (Figs. 3A–D, 4), labeling washeavy within: 1) the medial frontal polar (FPm), prelimbic(PL), medial orbital (MO), ventral orbital (VO), ventrolat-eral orbital (VLO), and anterior piriform (PIR) cortices(Fig. 4B,D); 2) the dorsal tenia tecta (TTd; Fig 4B,D); and3) the claustrum (CLA). Labeling was present but lessdense in the anterior cingulate (AC) and medial (frontal)agranular (AGm) cortices and was prominent within layer1 of these cortical fields (Figs. 3C,D, 4A,C).

More caudally in the rostral forebrain (Fig. 3E–H), la-beling remained strong dorsoventrally within the mPFC,stronger ventrally in the infralimbic (IL) and prelimbiccortices than dorsally in AGm and AC, and heavily con-centrated in layers 1 and 5/6 of IL and PL (Figs. 3E–H,5A–C). Labeling was also pronounced within the ventralagranular insular cortex (AIv), IL, TTd, and CLA and lightto moderate within the lateral (frontal) agranular cortex(AGl), PIR, olfactory tubercle (OT), and medial parts of thestriatum (Fig. 3E–H). A dense cluster of (terminally) la-beled fibers was present in the rostral pole of nucleusaccumbens (ACC; Figs. 3F, 5A,C), but few fibers were seen

TABLE 1. Density of Labeling in Forebrain Produced by PHA-L Injections in the Reuniens and Rhomboid Nuclei of the Midline Thalamus1

Structures

Labeling

Structures

Labeling

RE RH RE RH

TelencephalonCortex Dentate gyrus — —

Cingulate �� ��� Subiculum ��� ���Ectorhinal �� �� Lateral septumEntorhinal ��� ��� Dorsal n. — —

Medial ��� — Intermediate n. � ��Lateral ��� �� Ventral n. � �

Frontal polar Lateral preoptic area � �Medial ��� ��� Magnocellular preoptic n. � �Lateral � � Medial preoptic area � �

Infralimbic ��� ��� Median preoptic n. � �Insular Medial septal n. � �

Dorsal agranular �� ��� Olfactory tubercle � ��Ventral agranular �� ��� Septofimbrial n. — —Posterior agranular � �� Septohippocampal n. — —Dysgranular � � Substantia innominata � ��Granular � � Tania tecta

Lateral agranular (motor) �� � Dorsal ��� ���Medial agranular (motor) �� �� Ventral � �Occipital — ��Orbital Diencephalon

Lateral �� � ThalamusMedial ��� ��� Anterodorsal n. — —Ventral ��� �� Anteromedial n. � �Ventrolateral � � Anteroventral n. � �

Perirhinal ��� ��� Central lateral n. — —Piriform Central medial n. � �

Anterior part ��� �� Interanteromedial � �Posterior part � � Intermediodorsal n. � �

Prelimbic ��� ��� Lateral geniculate n. — —Retrosplenial ��� ��� Lateral habenula � �Somatosensory I � �� Llaterodorsal n. — —Somatosensory II � �� Lateroposterior n. — —Temporal � �� Medial geniculate n. — —

Accumbens n. Medial habenula — —Shell � ��� Mediodorsal n.Core � ��� Medial division � �

Amygdala Central division � �Anterior area — � Lateral division — —Basolateral � ��� Paracentral n. — —Basomedial � �� Parafascicular n. — —Central Paratential n. � —

Capsular part — � Paraventricular n.Medial part — � Anterior part � �

Cortical Posterior part � �Anterior part — � Posterior n. — —Posterior part Reticular n. � �

Medial � — Reuniens n. � �Lateral � � Rhomboid n. � �Posterior � � Submedial n. � �

Anterior olfactory n. Ventral anterior-lateral n. — —Medial part � � Ventral basal copmplex — —Ventral part � �� Hypothalamus

Bed n. of stria terminalis � �� Anterior n. — —Caudate-putamen � �� Dorsal hypothalamic area — —Claustrum ��� ��� Dorsomedial n. — —Diagonal band n. Lateral n. � �

Horizontal limb � � Mammillary bodies � —Vertical limb � � Paraventricular n. — —

Endopiriform n. — �� Posterior n. — �Globus pallidus — — Supramammillary n. � �Hippocampal formation Subthalamus

CA1 ��� ��� Zona incerta � �

1�, Light labeling; ��, moderate labeling, ���, dense labeling; —, absence of labeling; n, nucleus; PHA-L, Phaseolus vulgaris-leucoagglutinin; for other abbreviations see list.

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in other regions of ACC (Figs. 3G–K, 5B). For the mostpart, the labeling of the dorsal striatum (CP) involvedfibers of passage to the anterior forebrain. There was a

virtual absence of labeling in lateral regions of theprefrontal/frontal cortex within the primary motor (AGl)and sensory/somatosensory cortices.

Fig. 3. Schematic representation of labeling present in select sections through the forebrain anddiencephalon (A–U) and ventral hippocampus (AA–II) produced by a PHA-L injection (dots in Q–S) innucleus reuniens (case RE-20). Sections modified from the rat atlas of Swanson (1998). For abbreviationssee list.

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774 R.P. VERTES ET AL.

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Figure 3 (Continued)

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Labeling within midlevels of the forebrain (anterior

septum to rostral hippocampus; Fig. 3I–P). At midlev-els of the anterior forebrain (Fig. 3I–P), labeling waslargely confined to cortical sites, TTd, and claustrum.Within the cortex (Fig. 3I–L), relatively significant num-bers of labeled fibers were present: 1) dorsomedially in ACand AGm and, as rostrally, heavily concentrated in layers1, 5, and 6 of these fields and 2) ventrolaterally in theinsular cortex, extending from the dorsal and ventralagranular insular fields (AId and AIv) caudally to theposterior agranular insular cortex (AIp). Labeled fibersspread quite uniformly throughout all layers of the insularcortex. In addition, AGl (primarily outer layers) was mod-erately labeled, and parts of the basal forebrain, including

the medial and lateral septum and diagonal band nuclei,were lightly labeled.

More caudally (Fig. 3M–P), labeled fibers were localizedmainly to specific dorsomedial and ventrolateral parts ofthe cortex and to CLA, with few present in other regions ofcortex or subcortically. Specifically, labeling was pro-nounced dorsomedially in AC and AGm, rostrally (Fig.3M–O) and in the anterior pole of the retrosplenial cortex(RSC; Fig. 3P) caudally, as well as ventrolaterally in AIp.The labeled fibers lateral to AGm/RSC within AGl and thesomatosensory cortex, largely confined to layer 1 of thelateral convexity of cortex, appeared mainly to traversethese regions in route to parahippocampal cortices. Sub-cortically, the magnocellular preoptic nucleus, lateral pre-

Figure 3 (Continued)

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Fig. 4. A,B: Low-magnification darkfield photomicrographs oftransverse sections through the rostral forebrain showing patterns oflabeling within the orbitomedial prefrontal cortex produced by aninjection in nucleus reuniens (case 20). C,D: High-magnification dark-field photomicrographs taken from B (arrowheads). Note the presence

of pronounced numbers of labeled fibers along the medial wall of themedial prefrontal cortex (densely concentrated in layers 1 and 5/6) aswell as in the dorsal tenia tecta and the anterior piriform cortex (C,D)and the general absence of labeling in lateral regions of the cortex. Forabbreviations see list. Scale bar � 1,000 �m for A,B; 250 �m for C,D.

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Fig. 5. A,B: Low-magnification darkfield photomicrographs oftransverse sections through the rostral forebrain showing patterns oflabeling within the orbitomedial prefrontal cortex produced by aninjection in nucleus reuniens (case RE-20). Note the presence of pro-nounced numbers of labeled fibers in the infralimbic (IL), prelimbic(PL), anterior cingulate (AC), and medial (frontal) agranular cortices,stronger in IL and PL than AC and AGm, as well as in the claustrum,

dorsal tenia tecta, rostral pole of nucleus accumbens, and ventralagranular insular cortex. C: High-magnification darkfield photomi-crograph from A (arrowheads) showing labeled fibers in all layers ofIL, PL, and AC, densely concentrated in layers 1 and 5/6, and amediolateral orientation within middle layers of these fields parallelto the cell layers. For abbreviations see list. Scale bar � 1,000 �m forA,B; 250 �m for C.

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optic area, paraventricular nucleus of thalamus, and lat-eral hypothalamus were lightly labeled.

Labeling within posterior levels of the forebrain

(from anterior hippocampus to caudal diencephalon;

Fig. 3Q–U). As was found rostrally (Fig. 3E–P), labelingwithin the (iso-/allo-) cortex was confined mainly to dorso-medial and ventrolateral parts of cortex; that is, to thedorsal and lateral agranular RSC (RSCd and RSCagl;Risold et al., 1997) dorsomedially, and to the perirhinalcortex (PRC; particularly inner layers) and to lesser de-gree to the ectorhinal (ECT) and the lateral entorhinal(EC) cortices, bordering PRC, ventrolaterally. Labeled fi-bers on the lateral convexity of cortex (Fig. 3Q–U) ap-peared largely bound for ECT, PRC, and EC. At thesesame levels (Fig. 3R–U), a dense band of labeled fibers waspresent throughout stratum lacunosum-moleculare (slm)of CA1 of the dorsal hippocampus (Figs. 3R–U, 6A). Therewas an absence of labeling in CA2 or CA3 of Ammon’shorn or in the dentate gyrus (Fig. 6A). Subcortically, themediodorsal and central medial nuclei of thalamus, thesupramammillary nucleus of the hypothalamus and themedial, anterior cortical, lateral, and basolateral nuclei ofamygdala were lightly labeled.

Labeling with the ventral hippocampus (Fig. 3AA–

HH). As discussed, labeled fibers reached the ventralhippocampus, subiculum, and parahippocampal regionsmainly through the cingulum bundle (CB; Figs. 3AA,BB,6A,B) and secondarily through lateral cortical routes, thatis, through the dorsomedial PFC, around the lateral con-vexity of cortex (mainly within layer 1) to ventrolateralregions of cortex and parts of the subiculum. As depictedin Figure 3AA–HH, the ventral hippocampus and associ-ated parahippocampal regions were densely labeled. La-beled fibers were abundantly present throughout the slmof CA1, the dorsal and ventral subiculum, the pre- andparasubiculum, and the ECT, PRC and lateral EC (ECl;Fig. 3AA–HH). As seen with the dorsal hippocampus (Fig.3R–U), the entire extent of slm of CA1 of the ventralhippocampus was heavily labeled (Fig. 3AA–HH). This isdepicted at two levels of the ventral hippocampus in thephotomicrographs in Figure 6B,C. Labeling was equallypronounced throughout the molecular layer of the ventralsubiculum, continuous caudally with slm of CA1 (Figs.3DD–FF, 6C), and significant but somewhat less dense inthe pre- and parasubiculum (heaviest in layer 1) and innerlayers of the postsubiculum (Figs. 3DD–HH, 7A).

Within the parahippocampus, dense collections of la-beled fibers were observed rostrocaudally throughout ECTand PRC, mainly confined to the region around the rhinalfissure, rostrally (Fig. 3R–FF), with some extension todorsal regions of ECT, caudally (Fig. 3GG,HH). Althoughstrongest in layer 1, labeling was present throughout alllayers of ECT and PRC (Figs. 6C, 7A). Within the EC,labeling was considerably stronger 1) in the ECl than themedial EC (ECm), 2) in the dorsal than the ventral divi-sions of ECl, and 3) in the caudal than the rostral parts ofthe dorsal ECl. Specifically, beginning approximately atthe level of the fusion of the dorsal and ventral CA1 of thehippocampus (Fig. 3BB) and continuing throughout thecaudal extent of EC, the entire expanse (all layers) of thedorsal ECl was densely labeled (Figs. 3AA–HH, 6B,C, 7A).

Injections in approximately the caudal two-thirds of RE(present case) produced dense labeling in the dorsal ECl, butlittle in the ventral ECl or the ECm (Figs. 2F–H, 6B,C, 7A);the reverse was true for rostral RE injections: considerably

stronger labeling in ECm and in the ventral ECl (EClv) thanin the dorsal ECl. This is depicted in Figure 7B–D, showingdense aggregates of labeled fibers in ECm and EClv at threelevels of ventral hippocampus produced by a rostral REinjection (case aRE 9, Fig. 2E,F). This contrasts with anessential absence of ECm and EClv labeling (Fig. 7A) with acaudal RE injection (Figs. 1A, 2F–H).

Nucleus perireuniens

Extending laterally from the main body of RE, for approx-imately the caudal half of RE, are the “lateral wings” or theperireuniens region, or nucleus perireuniens (pRE; Risold etal., 1997; Van der Werf et al., 2002). Injections within pREgave rise to a pattern of labeling similar to that observedwith injections in RE (proper), with some significant differ-ences. As expected, a notable difference was that labelingwith pRE injections was predominantly unilateral (ipsilat-eral), as opposed to evenly distributed on both sides of thebrain with RE (proper) injections. At best, a few labeledfibers were observed contralaterally with pRE injections inventrolateral regions of the cortex and in CA1 and subicu-lum of HF. Similar to RE, labeling was more pronouncedcortically than subcortically and was restricted primarily to“limbic” regions of cortex, including the orbitomedial, insular(anterior and posterior divisions), ECT, PRC, EC (mainlyECl), and CA1/subiculum of HF. For the most part, fewerlabeled fibers were observed in commonly labeled sites withpRE than with RE injections. This was particularly the casesubcortically within ACC and CLA. A few regions, however,showed stronger labeling with pRE compared with RE injec-tions. The most notable of these were the ventral and ven-trolateral orbital cortices (VO, VLO), ECl, and ventral sub-iculum. Specifically, layers 1 and 3 of VO and VLO and tolesser extent LO (Fig. 8A), the longitudinal extent of EClimmediately ventral to the rhinal fissure (Fig. 8B,C), and theventral subiculum (Fig. 8B,C) were densely labeled withpRE injections.

Rhomboid nucleus: case RH-7

The site of injection for the schematically illustrated RHcase (case RH-7) is shown in Figure 1C,D. As depicted,labeled cells are localized to midlevels of RH (Figs. 1C,D,2G,H). Figure 9 schematically depicts the patterns of dis-tribution of labeled fibers after this injection. Similar toRE cases, the bulk of labeled fibers coursed ventrolaterallyfrom RH (Fig. 9P–R) and at the exit level from RH, splitinto two main bundles: one ascending to the rostral fore-brain in the general region of the MFB, the other coursinglaterally to parts of the amygdala and parahippocampalcortices. At the caudal septum, some labeled axons of theascending bundle continued forward to parts of the basalforebrain (Fig. 9F–N); the majority, however, turned dor-solaterally into the striatum to join the internal capsule,coursing medially/dorsomedially through the striatum tothe anterior forebrain. At the rostral forebrain, fibers ofthis bundle spread terminally to regions of the frontalcortex or swung caudally coursing within the cingulumbundle or through lateral parts of the cortex to posteriorregions of cortex and to the hippocampus. The secondarybundle exited laterally from RH, primarily bound for theamygdala, parahippocampal cortices, and ventral subicu-lum.

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Fig. 6. Low-magnification darkfield photomicrographs showingpatterns of labeling in the dorsal (A) and ventral (B,C) hippocampusproduced by an injection in nucleus reuniens (case RE-20). Note thedense concentration of labeled fibers restricted to the stratum lacu-nosum moleculare of CA1 of the dorsal (A) and ventral (B) hippocam-

pus and the molecular layer of the ventral subiculum (C). Note alsopronounced labeling in the perirhinal cortex (mainly layer 1) and the(dorsal) lateral entorhinal cortex. For abbreviations see list. Scalebar � 600 �m for A; 1,000 �m for B,C.

The Journal of Comparative Neurology. DOI 10.1002/cne

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Fig. 7. Low-magnification darkfield photomicrographs throughthe ventral hippocampus comparing patterns of labeling in the ento-rhinal cortex (EC) produced by a caudal (case RE-20; A) and rostral(case aRE-9; B–D) injection in nucleus reuniens. Note the caudal REinjection gave rise to labeling essentially confined to the (dorsal)lateral EC (ECl), whereas, by contrast, the rostral injection produced

by pronounced labeling within the ventral division of ECl (EClv) andin the medial entorhinal cortex (ECm) at three levels of the ventralhippocampus (B–D). Note also the presence of substantial labelingwith the rostral RE injection in the ventral subiculum, presubiculumand parasubiculum. For abbreviations see list. Scale bar � 1,000 �m.

The Journal of Comparative Neurology. DOI 10.1002/cne

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Fig. 8. Low-magnification darkfield photomicrographs of trans-verse sections through the anterior forebrain (A) and ventral hip-pocampus (B,C) showing patterns of labeling produced by an injectionin the perireuniens nucleus (case pRE-1). Note dense labeling in the

medial, ventral, and ventrolateral orbital cortices of the prefrontalcortex (A) as well as in the ventral subiculum and lateral entorhinalcortex at two levels of the ventral hippocampus (B,C). For abbrevia-tions see list. Scale bar � 750 �m for A; 1,000 �m for B,C.

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Anterior levels of the forebrain (preseptum; Fig. 9A–

G). At the anterior forebrain (Fig. 9A–G) labeled fiberswere concentrated in medial and ventrolateral regions ofthe prefrontal/frontal cortex as well as within the dorsal

and ventral striatum. Specifically, labeled axons spreadwidely throughout mPFC to the medial frontal polar(FPm), PL, AC and medial orbital (MO) cortices (Fig.9A–D). Labeling was heaviest in inner layers (5/6) of FPm,

Fig. 9. Schematic representation of labeling present in select sec-tions through the forebrain and diencephalon (A–U) and ventral hip-pocampus (AA–II) produced by a PHA-L injection (dots in P–R) in the

rhomboid nucleus (case RH-7). Sections modified from the rat atlas ofSwanson (1998). For abbreviations see list.

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783EFFERENTS OF REUNIENS AND RHOMBOID NUCLEI

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Figure 9 (Continued)

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Figure 9 (Continued)

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785EFFERENTS OF REUNIENS AND RHOMBOID NUCLEI

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PL, and MO (Fig. 10A) but extended uniformly to alllayers of AC. Although present, considerably fewer labeledfibers were observed in VO, VLO, anterior piriform cortex,and claustrum (Fig. 9A–D), and only trace amounts wereseen laterally in the lateral frontal polar (FPl) and dorsalagranular insular cortices (AId).

More caudally in the anterior forebrain (Fig. 9E–G),labeling remained pronounced along the inner wall ofmPFC, spreading fairly evenly across all layers of AC andAGm, but much denser in inner (5/6) than outer layers(1–3) of PL and IL (Fig. 9E–G). Similar to RE, labeledfibers in layers 2/3 of AC, PL, and IL were oriented pre-dominantly mediolaterally, parallel to cell layers of theseregions. Dense collections of labeled axons were also ob-served within the ventral striatum (nucleus accumbens)and adjacent ventromedial parts of the dorsal striatum(CP; Fig. 10B,C). As depicted (Fig. 9F,G), beginning at itsanterior pole, the entire rostrocaudal extent of ACC washeavily labeled (Fig. 9F–J). Although labeling was stron-ger in the core than in the shell of ACC, both regions weredensely labeled, particularly the core region adjacent tothe anterior commissure (see Fig. 10B,C). Although themajority of labeled fibers within the ventrolateral stria-tum (CP) appeared bound for the anterior forebrain, asignificant percentage terminated in CP (Fig. 10B,C).Other moderately to heavily labeled sites (Fig. 9E–H)were the olfactory tubercle (Fig. 10B), CLA, and AId/AIv.

Midlevels of the forebrain (anterior septum to ros-

tral hippocampus; Fig. 9H–O). At anterior levels of theseptum (Fig. 9H–K), labeling was largely confined to dor-somedial and ventrolateral regions of cortex and to thenucleus accumbens/olfactory tubercle. Within the dorso-medial cortex, significant numbers of labeled fibers werepresent in AGm, capping the cingulum bundle, mostheavily in layers 1 and 5/6. The medially adjacent AC wasless prominently labeled. On the ventrolateral convexityof cortex (Fig. 9H–K), labeled axons, bordering the exter-nal capsule, were found within CLA and to a lesser extentin the endopiriform nucleus (EN). The entire traverse ofCLA was strongly labeled (Fig. 9C–O). Lateral to CLA, theinsular cortex, extending from AIv to AIp, was moderatelylabeled. As seen rostrally, labeled fibers blanketed caudalparts of ACC and also spread heavily to the ventrallyadjacent OT. Apart from light to moderate labeling in theventromedial CP (mainly fibers of passage) and the lateralseptum, remaining regions of the basal forebrain weresparsely labeled. This included the medial, lateral, andmagnocellular preoptic nuclei; diagonal band nuclei; andsubstantia innominata (SI).

More caudally within the basal forebrain (Fig. 9L–O),labeling was largely confined to dorsomedial and ventro-lateral regions of cortex. Although significant numbers oflabeled axons continued to be present in AGm (all layers)and in CLA, they thinned considerably in AIp. Subcorti-cally, modest numbers of labeled fibers located within thebed nucleus of stria terminalis (BST), SI, and medial re-gions of basal forebrain/rostral diencephalon were des-tined mainly for the anterior forebrain (Fig. 9L–O).

Posterior levels of the forebrain (anterior hippocam-

pus to the caudal diencephalon; Fig. 9P–U). Similar torostral levels (Fig. 9A–P), labeling at the caudal forebrain(Fig. 9P–U) was restricted primarily to the (neo-) cortex,hippocampus, CLA, and amygdala and was negligibleelsewhere. Within the cortex, a continuum of labeled fi-bers of the dorsomedial cortex, beginning rostrally in

AGm/AC, extended caudally to the dorsal retrosplenialcortex (RSCd), lateral/dorsolateral to CB (Fig. 11A). Al-though present throughout all layers, they were mostheavily concentrated in inner layers (5/6) of RSCd. Theprimary somatosensory cortex (SSI), rostrally, and theposterior parietal cortex (PPC), caudally, lateral to RSCd,were moderately labeled. Some of this labeling, however,as well as that continuous with it within special sensoryregions of cortex (Fig. 9R–U), represents fibers bound forthe hippocampus/parahippocampus (see below).

Labeled fibers continued to occupy ventrolateral regionsof cortex, localized to AIp, rostrally (Fig. 9P–R), and toectorhinal and perirhinal cortices, caudally (Figs. 9S–U,11B). Labeling was moderate and largely confined to innerlayers of AIp, ECT, and PRC. Although fewer than seenwith RE injections, moderate numbers of labeled fiberswere present in the dorsal hippocampus, forming a tightband in slm of CA1 (Fig. 11A). Subcortically, labeling wasvirtually restricted to the amygdala. The basolateral andbasomedial nuclei were moderately to densely labeled(Figs. 9P–T, 11B), the medial and lateral posterior corticalnuclei lightly labeled. There was a virtual absence of la-beling throughout the diencephalon, both in the hypothal-amus and in the thalamus.

Ventral hippocampal region (Fig. 9AA–II). At cau-dal levels of the hippocampus (Fig. 9AA–II), labeling wasconfined to the hippocampal formation (CA1 and parts ofthe subiculum) and adjacent regions of cortex: RSC, occip-ital cortex (OC), ECT, PRC, and EC. As with the dorsalhippocampus, a narrow band of labeled fibers was presentwithin the outer molecular layer of the dorsal subiculum/dorsal CA1 (Fig. 9AA–CC). Unlike that of RE, this label-ing did not extend dorsoventrally throughout CA1/ventralsubiculum of the rostroventral hippocampus (see Fig.9AA–CC) but was restricted to dorsal aspects of HF (seeFig. 9AA–CC). This is depicted in the photomicrograph inFigure 12A. More caudally, however, labeled fibers of thedorsal subiculum joined those of the ventral subiculum, toform a continuous strip within the molecular layer ofsubiculum of the ventral HF (Figs. 9DD–GG, 12,B,C). Aswith the subiculum, the presubiculum (layer 1) washeavily labeled (Figs. 9GG–II, 12C,E); the para- and post-subiculum were lightly to moderately labeled.

In contrast to RE, caudal levels of the retrosplenialcortex were densely labeled, particularly the lateralagranular RSC. As depicted (Fig. 9AA,BB), sizeable num-bers of labeled fibers were present dorsal/dorsolateral tothe splenium of corpus callosum, mainly within RSCd andRSCagl, and continued to occupy this same positionthroughout the caudal extent of RSC (Fig. 9AA–II, 12A–C,E). Although fewer than in RSC, significant numberswere also found in the dorsal occipital area (Fig. 12C,E),ventral to the retrosplenial cortex. Some of these, how-ever, appeared bound for ECT, PRC, and ECl (Fig. 9CC–II). Among parahippocampal sites (ECT, PRC, EC), label-ing was densest within layers 1, 5/6 of ECT and layers 1and 4–6 of the ventrally adjacent ECl (Figs. 9DD–II,12A–D). Caudally, the pronounced labeling on the lateralconvexity of cortex (Fig. 9GG–II) variously in OC andposterior parietal and ventral temporal cortices appearedmainly terminal.

Central medial nucleus

For comparisons with injections in RE and RH, controlinjections were made in the CEM of thalamus, located

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Fig. 10. A,B: Low-magnification darkfield photomicrographs oftransverse sections through the anterior forebrain showing patternsof labeling in the medial prefrontal cortex (mPFC) and the dorsal andventral striatum produced by an injection in the rhomboid nucleus(case RH-7). Note dense labeling in inner layers (5/6) of the medialorbital and prelimbic cortices of mPFC (A) as well as in the nucleus

accumbens (ACC) and adjacent ventromedial parts of the dorsal stri-atum. C: High-magnification darkfield photomicrograph from B (ar-rows) showing substantial numbers of labeled fibers in the core andshell of ACC, particularly in the core of ACC, dorsal and lateral to theanterior commissure, as well as in ventrolateral dorsal striatum. Forabbreviations see list. Scale bar � 750 �m for A,B; �m 400 �m for C.

The Journal of Comparative Neurology. DOI 10.1002/cne

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Fig. 11. Low-magnification darkfield photomicrographs of trans-verse sections through dorsal (A) and ventrolateral (B) regions of theforebrain showing labeling produced by an injection in the rhomboidnucleus (case RH-7). Note dense labeling within stratum lacunosummoleculare of CA1 of the dorsal hippocampus and within several

regions of cortex, including the retrosplenial, primary, and secondarymotor and primary somatosensory cortices, dorsally (A), as well aswithin the basal lateral nucleus of the amygdala and parahippocam-pus (ectorhinal, perirhinal, and entorhinal cortices), ventrally (B). Forabbreviations see list. Scale bar � 800 �m for A; 1,000 �m for B.

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Fig. 12. A–C: Low-magnification darkfield photomicrographsshowing patterns of labeling at three levels of the ventral hippocam-pus produced by an injection in the rhomboid nucleus (case RH-7).Note pronounced labeling in the dorsal subiculum/dorsal CA1 of therostroventral hippocampus but not in the ventral subiculum/ventralCA1 at this level (A) and labeling in the ventral subiculum at caudallevels of the ventral hippocampus (B,C). Note also the substantial

labeling along the lateral convexity of cortex within the retrosplenial,occipital, and temporal cortices and particularly dense in layers 1 and5 of the ectorhinal and perirhinal cortices. D,E: High-magnificationdarkfield photomicrographs from C (arrowheads) showing heavy la-beling within the ectorhinal cortex (D) and in the occipital and tem-poral cortices and layer 1 of the postsubiculum (E). For abbreviationssee list. Scale bar � 1,250 �m for A–C; 750 �m for D; 500 �m for E.

The Journal of Comparative Neurology. DOI 10.1002/cne

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dorsal to RH. Although there was some overlap in pat-terns of labeling with CEM injections compared withRE/RH injections, differences predominated. Labelingproduced by CEM injections was virtually restricted to therostral forebrain; few labeled fibers were seen caudal tothe level of CEM. Within the anterior forebrain, labelingwas pronounced throughout FPm (all layers), extendingfrom its anterior tip to its juncture with AGm (Fig. 13A).The laterally adjacent FPl was lightly labeled with rostralCEM injections and moderately labeled with caudal CEMinjections.

Significant numbers of labeled axons occupied dorsome-dial regions of the cortex caudal to FPm within AGm andto a lesser extent in AC (Fig. 13A,B). There was a progres-sive decline in labeling at successive caudal levels of AGm/AC, continuing to the retrosplenial cortex, which was vir-tually devoid of labeled fibers. The rostrocaudal extent ofthe dorsal agranular insular cortex (AId) was denselylabeled (Fig. 13A,B). Considerably fewer labeled fiberswere present in the caudally adjacent AIp, with the excep-tion of caudal pole of AIp, bordering the ectorhinal cortex.A restricted strip of labeled fibers stretching across layer5 of AIp, ECT, and PRC was observed. Subcortically, la-beling was heavy within the dorsal and ventral (ACC)striatum, the olfactory tubercle, and the basolateral nu-

cleus (BLA) of the amygdala. Massive numbers of labeledfibers were present within ACC (bordering the anteriorcommissure) as well as within ventrolateral/lateral re-gions of CP, rostrally, and the entire lateral two-thirds ofCP, caudally. This labeling as well as that of the adjacentolfactory tubercle is depicted at two levels of the forebrainin the photomicrographs in Figure 13. Amygdaloid label-ing was pronounced within, and was essentially restrictedto, anterior and posterior divisions of BLA (Fig. 14A,B).

DISCUSSION

We examined, compared, and contrasted the efferentprojections of nucleus reuniens (RE) and the rhomboidnucleus (RH) of the midline thalamus. Main RE targetsare the prefrontal cortex, parahippocampal cortex (ecto-rhinal, perirhinal, and entorhinal cortices), and hippocam-pal formation. RH distributes more widely than RE, thatis, to most RE projection sites, but also to somatosensory,posterior parietal, retrosplenial, temporal, and occipitalcortices as well as to the nucleus accumbens and basolat-eral nucleus of amygdala. RE and RH projections differfrom those of the CEM of thalamus. In common with RH,CEM distributes to nucleus accumbens, OT, and BLA.Unlike RE/RH, CEM sends few fibers to limbic and sen-

Fig. 13. Low-magnification darkfield photomicrographs of trans-verse sections through the rostral forebrain showing patterns of la-beling produced by an injection in the central medial nucleus of themidline thalamus (case CEM-10). Note dense labeling in dorsalagranular insular cortex (A), the core and shell of nucleus accumbens

(ACC), the olfactory tubercle (A,B), and the dorsal striatum (lateral/dorsolateral to ACC; B) and moderate labeling in the medial (frontal)agranular cortex (A,B). For abbreviations see list. Scale bar � 1,000�m.

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sory regions of cortex and none to the hippocampus, pro-jecting instead to the motor cortex and dorsal striatum.

There is a shift in patterns of projections from ventral todorsal regions of the ventral midline thalamus such thatmain targets: 1) of RE are limbic cortex and hippocampus;2) of RH are limbic and sensorimotor cortices, hippocam-pus, ACC, OT, and BMA/BLA; and 3) of CEM are motorcortex, dorsal striatum, and subcortical sites innervatedby RH. In effect, the ventral-to-dorsal gradient in projec-tions represents a transition from virtually solely limbic(RE), to sensorimotor/limbic (RH), to largely motor/limbic(CEM). Overall, the ventral midline thalamus is strategi-cally positioned to exert significant control over fairlywidespread regions of the cortex (limbic, sensory, motor),the hippocampus, the dorsal and ventral striatum, and thebasal nuclei of the amygdala, possibly involved in coordi-nating limbic and sensorimotor functions.

Summary of RE projections andcomparisons with previous studies

The primary prefrontal/frontal targets of RE were themedial frontal polar cortex (FPm), the medial and ventralorbital cortices, AGm, AC, PL (anterior and posterior di-visions), and IL (of mPFC), and the dorsal, ventral andposterior agranular insular cortices. The main RE targetsoutside of the prefrontal cortex (PFC) were the rostralretrosplenial cortex and parahippocampus/hippocampus.As described, RE distributes massively to the parahip-pocampus and HF; that is, to the perirhinal and entorhi-nal cortices, the slm of CA1 of the dorsal and ventralhippocampus, the molecular layer of the dorsal and ven-tral subiculum, and the parasubiculum and significantlybut somewhat less densely to the ectorhinal (postrhinal)cortex and the pre- and postsubiculum. There was anabsence of RE projections to CA2 and CA3 fields of Am-mon’s horn and to the dentate gyrus. The rostral REdistributes more heavily to the medial EC than to thelateral EC, whereas the caudal RE (caudal two-thirds ofRE) projects more densely to the lateral (dorsal division ofECl) than to the medial EC.

Although RE projections to the hippocampus have beenwell described (Herkenham, 1978; Wouterlood et al., 1990;Wouterlood, 1991; Dolleman-Van der Weel and Witter,1996; Bokor et al., 2002), few reports have examined over-all patterns of RE projections (Herkenham, 1978; Ohtakeand Yamada, 1989; Van der Werf et al., 2002). Perhapsthe most complete analysis of RE projections was an earlystudy by Herkenham (1978) using the autoradiographictechnique. Followup studies, using newer tracers, haveconcentrated on RE projections to the hippocampus (Wouter-lood et al., 1990) and to the EC (Wouterlood, 1991) or pro-jections from specific regions of RE (Risold et al., 1997).

In general, the findings of previous studies support thepresent results. With some exceptions, there appears to beagreement that RE targets mainly “limbic” (neo/allo) cor-tex and the hippocampus and to a considerably lesserdegree subcortical structures. In partial contrast to earlierreports, however, we observed considerably stronger pro-jections to commonly labeled regions (particularly PFC,dorsal hippocampus, and ECl) and projections to a numberof sites not previously described. Consistent with earlierfindings, we showed that RE distributes significantly tothe mPFC and more heavily to IL/PL than to AC/AGm(Herkenham, 1978; Wouterlood et al., 1990; Risold et al.,1997), but unlike these studies, we further demonstrated

that RE projects substantially to the anterior pole of thePFC (rostral to the genu of CC), that is, to FPm, anteriorPL and AC, orbital cortices (MO, VO, VLO), dorsal andventral agranular insular cortices, and anterior piriformcortex. In accord with the findings of Van der Werf et al.(2002), we showed that the orbital cortex receives partic-ularly dense projections from pRE.

Previous studies have described relatively massive REprojections to the ventral hippocampus but at best lightones to the dorsal hippocampus, leading to the conclusionthat RE distributes to the ventral but not dorsal HF(Herkenham, 1978; Ohtake and Yamada, 1989; Su andBentivoglio, 1990; Wouterlood et al., 1990; Dolleman-Vander Weel and Witter, 1996; Risold et al., 1997). For in-stance, following their findings of modest RE projectionsto the dorsal hippocampus, Risold et al. (1997) suggestedthat previous demonstrations of stronger projections(Wouterlood et al., 1990) probably resulted from thespread of injections to the overlying rhomboid nucleus.Although RH distributes to the dorsal hippocampus (Be-rendse and Groenewegen, 1991, present results), the pro-jections are more restricted and less robust than thosefrom RE.

In contrast to our results, some reports (Herkenham,1978; Ohtake and Yamada, 1989) but not others (Wouter-lood et al., 1990; Risold et al., 1997) have described fairlywidespread RE projections to several subcortical sites,including the nucleus accumbens, OT, septum, bed nu-cleus of stria terminalis, medial and lateral preoptic areas,basal nuclei of amygdala, median eminence (ME), lateralhypothalamus, ventral tegmental area, pretectum, supe-rior colliculus, and midbrain central gray. With the excep-tion of projections to the rostral pole of ACC, and some toOT and LS, we failed to observe (terminal) labeling in anyof these structures. Wouterlood et al. (1990) similar failedto detect such labeling and argued that earlier descrip-tions probably resulted from the inclusion of parts of thehypothalamus, particularly the paraventricular nucleus ofhypothalamus, in the injections. Consistent with this, weobserved moderate, and in some cases fairly dense, label-ing in many of the above-mentioned structures with injec-tions that spread ventrally to the paraventricular nucleusor to the posterior nucleus of the hypothalamus (see alsoVertes et al., 1995).

Although previous reports have shown that RE heavilytargets EC, studies differ in their description of precisepatterns of innervation of EC, that is, strong projections toECl and weak ones to ECm (Herkenham, 1978), the re-verse (ECm � ECl; Risold et al., 1997), or essentially equaldensity (Wouterlood et al, 1990). The present demonstra-tion that the rostral RE preferentially distributes to ECmand the caudal RE to ECl may explain these differences.

Summary of RH projections andcomparisons with previous studies

The major cortical projection sites of RH are the FPm,medial orbital cortex, mPFC, retrosplenial cortex, poste-rior parietal cortex, occipital cortex, parahippocampus,and HF. The main subcortical sites are the claustrum,ACC, OT, LS, and basal nuclei of the amygdala (BLA andBMA).

Similar to RE, RH fibers distribute widely over thePFC/frontal cortex, terminating heavily in FPm, medialorbital cortex, AGm, AC, PL, and IL (of mPFC) and mod-erately in the lateral frontal polar, the anterior piriform,

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Fig. 14. Low-magnification darkfield photomicrographs of trans-verse sections through the forebrain showing patterns of labeling inthe amygdala produced by an injection in the central medial nucleus(case CEM-10). Note very dense labeling essentially confined to the

anterior (A) and posterior (B) divisions of the basolateral nucleus ofamygdala at these levels. For abbreviations see list. Scale bar � 500�m.

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ventral orbital, and insular (AId, AIv, AIp) cortices. Addi-tional cortical projection sites include caudal parts of AGmand AC and the retrosplenial, somatosensory, posteriorparietal, temporal, and occipital cortices. Although lesspronounced than RE, RH also distributes significantly tothe hippocampus and parahippocampus; that is, to slm ofCA1 of the dorsal and ventral hippocampus, the molecularlayer of the dorsal subiculum, the posteroventral subicu-lum, and the postsubiculum as well as to the perirhinalcortex (flanking RF) and inner layers (5/6) of the dorsalECl.

Few reports have examined the efferent projections ofRH (Ohtake and Yamada, 1989; Berendse and Groenewe-gen, 1991; Van der Werf et al., 2002). In general, thepresent findings are consistent with earlier demonstra-tions of a widespread distribution of RH fibers to thecortex and parts of the striatum. For instance, with regardto the cortex, Van der Werf et al. (2002) noted that, “incontrast to the other intralaminar and midline nuclei, theprojections of Rh are not confined to limbic structures andassociated cortices, but reach primary and secondary mo-tor and sensory cortices as well.” It is nonetheless clear,however, that RH distributes less densely to sensorimotorregions of cortex than to various “limbic” regions of cortex,including the hippocampus, suggesting that RH, like RE,exerts primary influence over subcortical/cortical limbicstructures.

Although previous reports have described RH projec-tions to HF (Yanaghihara et al., 1987; Su and Bentivoglio,1990; Berendse and Groenewegen, 1991; Van der Werf etal. 2002), they appear to be considerably less pronouncedthan shown here. For instance, Berendse and Groenewe-gen (1991) described relatively prominent RH projectionsto the dorsal subiculum and dorsal CA1 of the ventralhippocampus but failed to confirm our demonstration ofprojections to CA1 of the dorsal hippocampus and to theposteroventral subiculum. This may involve differing lo-cations of RH injections.

Consistent with the present results, Ohtake andYamada (1989) described prominent RH projections toACC, spreading virtually throughout ACC, whereas Be-rendse and Groenewegen (1991) reported that RH distrib-utes fairly selectively to the ventrolateral shell of ACC.Although RH fibers are more densely concentrated in thecore than in the shell of ACC (present results), the find-ings of several studies with retrograde tracers (Ohtakeand Yamada, 1989; Su and Bentivoglio, 1990; Brog et al.,1993; Otake and Nakamura, 1998), showing labeled cellsin RH with injections throughout ACC, support a ratherwidespread distribution of RH efferents to ACC.

In contrast to the present and earlier findings (Berendseand Groenewegen, 1991) that RH projects to limited sitesof the diencephalon and basal forebrain, Ohtake andYamada (1989) reported that RH distributes terminally toseveral subcortical structures, including the BST; the lat-eral, medial, basomedial, and basolateral nuclei of theamygdala; the anterior and lateral hypothalamus; thezona incerta; and the ventrolateral, ventromedial, ventro-posterior, central medial and submedial nuclei of thala-mus. With the exception of RH projections to BLA andBMA, we essentially failed to observe labeling in thesestructures (see also Berendse and Groenewegen, 1991).The injections of Ohtake and Yamada (1989) were largeand seemed to extend beyond the boundaries of RH, rais-

ing the possibility that their labeling originated from tha-lamic groups outside of RH.

Functional significance

Nucleus reuniens. We showed that the output of REis directed to the hippocampus, parahippocampus, andvarious regions of the prefrontal cortex. These “limbic”structures of cortex serve a well-recognized role in variousforms of memory processing (Baddeley, 1986, 1998; Dudai,1989; Goldman-Rakic, 1994, 1995; Eichenbaum et al.,1996; Eichenbaum and Cohen, 2001; Fuster, 2001; Verteset al., 2004a; Vertes, 2005). With the possible exception ofRH, no other nucleus of the thalamus displays a similarpattern of projections. Hence, RE is uniquely positioned toinfluence simultaneously major structures of the brain(HF and mPFC) subserving memory.

Although the output of RE is relatively restricted, REreceives a diverse and widely distributed set of afferentprojections, mainly from limbic and limbic-associatedstructures of the cortex, basal forebrain, hypothalamus,amygdala, and brainstem (Herkenham, 1978; Risold et al.,1997; Canteras and Goto, 1999; Krout et al., 2002; Olucha-Bordonau et al., 2003; McKenna and Vertes, 2004). Thissuggests that RE is a critical site for the convergence of adiverse array of limbic/limbic related information and itssubsequent transfer to its main targets, the hippocampus/parahippocampus and the orbitomedial PFC.

In previous examinations of the efferent projections ofthe mPFC in rats (Vertes, 2002, 2004), we showed that allfour divisions of mPFC (IL, PL, AC, and AGm) projectmassively to RE. Aside from RE, only the mediodorsalnucleus of thalamus receives similar projections (Groe-newegen, 1988; Sesack et al., 1989; Hurley et al., 1991;Price, 1995; Ongur and Price, 2000; Vertes, 2002; Gabbottet al., 2005).

Several reports for various species have demonstratedpronounced projections from the hippocampus to themPFC (Swanson, 1981; Irle and Markowitsch, 1982; Ca-vada et al., 1983; Goldman-Rakic et al., 1984; Ferino et al.,1987; Jay et al., 1989; van Groen and Wyss, 1990; Jay andWitter, 1991; Carr and Sesack, 1996), but there are noreturn projections from the mPFC to HF (Beckstead, 1979;Goldman-Rakic et al., 1984; Room et al., 1985; Reep et al.,1987; Sesack et al., 1989; Hurley et al., 1991; Takagishiand Chiba, 1991; Buchanan et al., 1994). For instance,Laroche et al. (2000) recently noted that “unlike otherneocortical areas such as the perirhinal or entorhinal cor-tices, which are reciprocally connected to the hippocampus(Witter et al., 1989), area CA1 and the subiculum do not,in return, receive direct projections from the prefrontalcortex in the rat.” The demonstration, then, of pronouncedmPFC projections to RE and similar dense RE projectionsto the hippocampus and EC, coupled with the absence ofmPFC to HF/EC projections, suggests that RE representsa critical relay in the transfer of information from themPFC to the HF/EC.

All cortical areas receive and send projections to thethalamus (Jones, 1985). The conventional view is that thecortical output to the thalamus primarily serves to mod-ulate return thalamocortical projections. Although, inpart, this is undoubtedly true, the paradoxical findings ofgreater than tenfold more corticothalamic than thalamo-cortical fibers suggests that cortical projections to thala-mus do not merely modulate return projections to cortex.In this regard, Llinas et al. (1998) proposed that the thal-

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amus may serve as a way station for intracortical commu-nication. Specifically, they suggested that the thalamusshould not be viewed simply as a gateway to the cortex;rather, “the thalamus represents a hub from which anysite in the cortex can communicate with any other suchsite or sites.”

Along these lines, we would suggest that the midlinethalamus (or RE and RH) might serve critically to inter-connect select cortical and subcortical limbic structures,that is, an important conduit (hub) in the transfer ofinformation among these structures. In contrast to directconnections between these structures (Groenewegen et al.,1990; Gabbott et al., 2003), information flow betweenthem, by way of RE, would appear subject to modulatoryinfluences acting on RE (McKenna and Vertes, 2004) thatmay serve to “gate” the transfer of information. In effect,depending on the type (or mode) of input it receives, REmay differentially channel information between specificsets (or subsets) of forebrain structures appropriate to thedemands of the behavioral situation, in a sense act as amaster switch.

Rhomboid nucleus. Similar to RE, RH may also servea critical role in intracortical and cortical-subcortical com-munication, but for a more diverse set of structures thanfor RE. In this regard, RH and RE receive similar sets ofafferents from the brainstem and hypothalamus butlargely different inputs from the forebrain. Specifically,reflecting its output, RH receives afferents from the limbicforebrain, but additionally pronounced projections fromprimary and secondary motor cortices (AGl and AGm) andthe primary somatosensory cortex (Vertes et al., 2004b;Owens, 2005). Based on its inputs and outputs, RH maybridge sensorimotor and limbic domains, possibly provid-ing limbic support (emotional/cognitive) for motor acts.

Integrated RE, RH, and CEM activity: the ventral

midline thalamus as a core component of the “limbic

thalamus.” The midline and intralaminar nuclei ofthalamus have been designated “nonspecific thalamus,”differentiating them from relay nuclei of the thalamus. Asdiscussed, the early notion that midline nuclei of thala-mus distribute widely throughout the cortical mantle andhence exert global effects on the cortex has been revised byfindings (Berendse and Groenewegen, 1991; Van der Werfet al., 2002; present results) showing that each of themidline nuclei exhibits a unique and restricted set of cor-tical (and subcortical) projections. With regard to theseprojections, we described a ventral-to-dorsal gradient inprojections of the ventral midline thalamus from almostentirely “limbic” (RE) to a progressively greater somatic/limbic mix (RH/CEM). The ventral midline thalamus isideally positioned to relay diverse, mainly limbic, informa-tion from various sources to forebrain structures involvedin emotional/cognitive and sensorimotor aspects of behav-ior.

Physiological effects of the ventral midline thalamus

on the hippocampus and mPFC. As described, majorRE targets are the hippocampus, EC, and mPFC. It hasrecently been shown stimulation of RE/RH producesstrong excitatory actions at CA1 of the hippocampus(Dolleman-Van der Weel et al., 1997; Bertram and Zhang1999), the EC (Zhang and Bertram, 2002), and the mPFC(Viana Di Prisco and Vertes, 2006). For instance,Dolleman-Van der Weel et al. (1997) showed that REstimulation produced 1) large negative-going field poten-tials at stratum lacunosum-moleculare of CA1 indicative

of a pronounced depolarizing action of RE on distal apicaldendrites of CA1 pyramidal cells and 2) a marked paired-pulse facilitation of evoked potentials at CA1. They pro-posed that RE may “exert a persistent influence on thestate of pyramidal cell excitability,” depolarizing cells toclose to threshold for activation by other excitatory inputs.

Consistent with this, Bertram and Zhang (1999) com-pared the effects of RE (midline thalamic) and CA3 stim-ulation on various population measures at CA1 andshowed that RE actions on CA1 were equivalent to, and insome cases considerably greater than, those of CA3 onCA1. They concluded that the RE projection to the hip-pocampus “allows for the direct and powerful excitation ofthe CA1 region. This thalamohippocampal connection by-passes the trisynaptic/commissural pathway that hasbeen thought to be the exclusive excitatory drive to CA1.”More recently, Zhang and Bertram (2002) similar demon-strated that RE stimulation produced short-term (evokedresponses) and long-term (paired-pulse facilitation andLTP) excitatory effects at EC and concluded that RE“plays a significant role in limbic physiology and mayserve to synchronize activity in this system.” Finally, werecently showed (Viana Di Prisco and Vertes, 2006) thatstimulation of the ventral midline thalamus (RE/RH) pro-duced 1) large-amplitude, monosynaptically elicitedevoked potentials dorsoventrally throughout the mPFC,with the largest effects elicited at the infralimbic (IL) andprelimbic (PL) cortices, and 2) pronounced paired-pulsefacilitation at IL and PL.

In summary, RE/RH target predominantly limbic fore-brain structures and exert pronounced excitatory actionson them. RE/RH are pivotally positioned to relay limbic(visceral, emotional) information to the forebrain, possiblyserving to control the flow of information among limbicsubcortical and cortical structures involved in complexbehaviors.

ACKNOWLEDGMENTS

We thank two anonymous reviewers for their excellentcomments on an earlier version of the manuscript. Wethank Balazs Szemes and William Jennings for graphicillustration work.

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