projections of the paraventricular and paratenial nuclei ...vertes/pv-pt.pdfprojections of the...

Projections of the Paraventricular andParatenial Nuclei of the Dorsal Midline

Thalamus in the Rat

ROBERT P. VERTES* AND WALTER B. HOOVER

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

ABSTRACTThe paraventricular (PV) and paratenial (PT) nuclei are prominent cell groups of the

midline thalamus. To our knowledge, only a single early report has examined PV projectionsand no previous study has comprehensively analyzed PT projections. By using the antero-grade anatomical tracer, Phaseolus vulgaris leucoagglutinin, and the retrograde tracer,FluoroGold, we examined the efferent projections of PV and PT. We showed that the outputof PV is virtually directed to a discrete set of limbic forebrain structures, including ‘limbic’regions of the cortex. These include the infralimbic, prelimbic, dorsal agranular insular, andentorhinal cortices, the ventral subiculum of the hippocampus, dorsal tenia tecta, claustrum,lateral septum, dorsal striatum, nucleus accumbens (core and shell), olfactory tubercle, bednucleus of stria terminalis (BST), medial, central, cortical, and basal nuclei of amygdala, andthe suprachiasmatic, arcuate, and dorsomedial nuclei of the hypothalamus. The posterior PVdistributes more heavily than the anterior PV to the dorsal striatum and to the central andbasal nuclei of amygdala. PT projections significantly overlap with those of PV, with someimportant differences. PT distributes less heavily than PV to BST and to the amygdala, butmuch more densely to the medial prefrontal and entorhinal cortices and to the ventralsubiculum of hippocampus. As described herein, PV/PT receive a vast array of afferents fromthe brainstem, hypothalamus, and limbic forebrain, related to arousal and attentive states ofthe animal, and would appear to channel that information to structures of the limbicforebrain in the selection of appropriate responses to changing environmental conditions.Depending on the specific complement of emotionally associated information reaching PV/PTat any one time, PV/PT would appear positioned, by actions on the limbic forebrain, to directbehavior toward a particular outcome over a range of outcomes. J. Comp. Neurol. 508:212–237, 2008. © 2008 Wiley-Liss, Inc.

Indexing terms: medial prefrontal cortex; subiculum of hippocampus; nucleus accumbens; bed

nucleus of stria terminalis; central and basal nuclei of amygdala

The paraventricular and paratenial nuclei are promi-nent cell groups of the midline thalamus (Swanson, 1998;Van der Werf et al., 2002). The paraventricular nucleus(PV) lies dorsally on the midline directly below the thirdventricle and extends rostrocaudally virtually throughoutthe thalamus. The paratenial nucleus (PT) borders PVlaterally and overlaps with approximately the rostral one-third of PV.

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) (Demp-sey and Morrison, 1942), the midline thalamus wasviewed as ‘nonspecific’ thalamus, exerting nonspecific or

global effects on the cortical mantle (Bentivoglio et al.,1991; Groenewegen and Berendse, 1994). The notion, how-ever, of the midline thalamus as ‘nonspecific’ has beenrevised based on the subsequent anatomical demonstra-

Grant sponsor: National Institute of Mental Health; Grant numbers:MH42900, MH63519.

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

Received 29 August 2007; Revised 20 December 2007; Accepted 10 Jan-uary 2008

DOI 10.1002/cne.21679Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 508:212–237 (2008)

© 2008 WILEY-LISS, INC.

tion that nuclei of the midline thalamus do not projectwidely throughout the neocortex, but rather selectively tospecific regions of cortex, primarily those of the prefrontalcortex (Berendse and Groenewegen, 1991; Van der Werf etal., 2002; Groenewegen and Witter, 2004; Vertes, 2006). Inaddition, recent reports have shown that stimulation ofindividual nuclei of the midline thalamus produce selec-tive effects on their cortical targets—as opposed to wide-spread actions throughout the cortex (Dolleman-Van derWeel et al., 1997; Bertram and Zhang, 1999; Kung andShyu, 2002; Zhang and Bertram, 2002; Viana Di Priscoand Vertes, 2006).

In a series of reports, Groenewegen and colleagues (Be-rendse and Groenewegen, 1990, 1991; Berendse et al.,1992; Groenewegen et al., 1999) showed that major tar-gets of midline thalamic nuclei were the prefrontal cortexand ventral striatum (nucleus accumbens, ACC), and fur-ther that recipient zones in the medial prefrontal cortex

(mPFC) and ACC were themselves directly connected(mPFC to ACC). With respect to the paraventricular nu-cleus, PV distributes to the prelimbic cortex (of mPFC)and to the medial shell region of ACC, and PL, in turn,projects to the shell of ACC (Berendse and Groenewegen,1990, 1991; Berendse et al., 1992).

Undoubtedly owing to the early emphasis on PV projec-tions to the ventral striatum and to mPFC, subsequentreports largely focused on these target sites. Using dualretrograde labeling techniques, Otake and Nakamura(1998) reported that of the nuclei of the midline thalamus,PV contained the largest percentage of cells with collat-eral projections to ACC and mPFC. In like manner, Bub-ser and Deutch (1998) showed that �15% of PV cellsdistribute via collaterals to the medial shell of ACC and PL,while Pinto et al. (2003) demonstrated that PV fibers termi-nate in close apposition to dopaminergic (DA) terminals innucleus accumbens, but not to DA terminals in the mPFC.

Abbreviations

AA Anterior amygdaloid areaac Anterior commissureAC Anterior cingulate cortexACC,c,s Nucleus accumbens, core and shell divisionsAGm Medial agranular (frontal) cortexAGl Lateral agranular (frontal) cortexAH Anterior nucleus of hypothalamusAI,d,p,v Agranular insular cortex, dorsal, posterior, ventral divi-

sionsAM Anteromedial nucleus of thalamusAON Anterior olfactory nucleusAV Anteroventral nucleus of thalamusBLA Basolateral nucleus of amygdalaBMA Basomedial nucleus of amygdalaBST Bed nucleus of stria terminalisCA1,3 Field CA1 and CA3 of Ammon’s horncc Corpus callosumCEA,c,l,m Central nucleus of amygdala, capsular, lateral, and me-

dial divisionsCL Central lateral nucleus of the thalamusCLA ClaustrumCM Central medial nucleus of thalamusCOA,a,p Cortical nucleus of amygdala, anterior, posterior divisionsCP Caudate-putamenDBh Nucleus of diagonal band, horizontal limbDMh Dorsomedial nucleus of hypothalamusEC,l Entorhinal cortex, lateral divisionECT Ectorhinal cortexEN Endopiriform nucleusfa Forceps of the corpus callosumFG FluorogoldFI Fimbria of hippocampusFP,l,m Frontal polar cortex, lateral, medial divisionsFS Fundus of striatumGI Granular insular cortexGP Globus pallidusHF Hippocampal formationIAM Interanteromedial nucleus of thalamusIL Infralimbic cortexIMD Intermediodorsal nucleus of thalamusIP Interpeduncular nucleusLA Lateral nucleus of amygdalaLD Lateral dorsal nucleus of thalamusLH Lateral habenulaLHy Lateral hypothalamusLO Lateral orbital cortexLP Lateral posterior nucleus of thalamusLPO Lateral preoptic areaLS Lateral septumMA Magnocellular preoptic nucleusMD Mediodorsal nucleus of thalamusMEA Medial nucleus of the amygdala

MFB Medial forebrain bundleMG Medial geniculate nucleus of thalamusMH Medial habenulaMO Medial orbital cortexmPFC Medial prefrontal cortexMPO Medial preoptic areaMPN Medial preoptic nucleusMRF Mesencephalic reticular formationMS Medial septummt Mammillothalamic tractOC Occipital cortexOT Olfactory tuberclePAp Posterior parietal cortexPC Paracentral nucleus of thalamusPFC Prefrontal cortexPH Posterior nucleus of hypothalamusPHA-L Phaseolus vulgaris-leucoagglutininPIR Piriform cortexPL Prelimbic cortexPO Posterior nucleus of thalamusPRC Perirhinal cortexPT Paratenial nucleus of thalamusPV,a,p Paraventricular nucleus of thalamus, anterior and poste-

rior divisionsRE Nucleus reuniens of thalamusRH Rhomboid nucleus of thalamusRN Red nucleusRSC Retrosplenial cortexRT Reticular nucleus of thalamusSC Superior colliculusSCN Suprachiasmatic nucleusSI Substantia innominatasm Stria medullarisSM Submedial nucleus of thalamusSNr Substantia nigra, pars reticulataSPZ Subparaventricular zone of hypothalamusSSI Primary somatosensory cortexSSII Secondary somatosensory cortexst Stria terminalisSUB,v Subiculum, ventral divisionSUM Supramammillary nucleusTE Temporal cortexTT,d,v Tenia tecta, dorsal and ventral divisionsV3 Third ventricleVAL Ventral anterior nucleus of thalamusVB Ventral basal nucleus of thalamusVL Lateral ventricleVLO Ventrolateral orbital cortexVM Ventral medial nucleus of thalamusVO Ventral orbital cortexVTA Ventral tegmental areaZI Zona incerta

The Journal of Comparative Neurology

213EFFERENTS OF PV AND PT NUCLEI

To our knowledge, only a single report (Moga et al.,1995) has examined the general distribution of PV projec-tions “with special emphasis on the projections to thehypothalamus and amygdala.” Focusing on circadian cir-cuitry, Moga et al. (1995) described PV projections to thesuprachiasmatic nucleus (SCN) as well as to other sitesinvolved in circadian rhythms including the dorsomedialnucleus and subparaventricular zone of the hypothala-mus. These results, coupled with the demonstration thatPV receives input from all major components of the circa-dian system including SCN, led Moga et al. (1995) toconclude that, “the anterior PV is ideally situated to relaycircadian timing information from the SCN to brain areasinvolved in visceral and motivation aspects of behaviorand to provide feedback regulation of the SCN.” Consis-tent with this, Peng and Bentivoglio (2004) recently dem-onstrated at the light and electron microscopic (EM) levelsthat SCN fibers synaptically contact PV cells projecting tothe amygdala and concluded that PV serves an importantrole in the “transfer of circadian timing information to thelimbic system.”

PV receives a vast array of afferents from monoaminer-gic and neuropeptide containing systems of the brainstemand hypothalamus—systems known to have activatingeffects on the forebrain (Chen and Su, 1990; Vertes, 1991;Freedman and Cassell, 1994; Bhatnagar et al., 2000;Krout et al., 2002; Otake, 2005). Accordingly, PV (andother midline thalamic nuclei) are thought to serve adirect role in processes of arousal and attention (Krout etal., 2002; Van der Werf et al., 2002; Vertes, 2002, 2006).Consistent with a role for PV in attention, Kirouac et al.(2005) recently showed that PV receives pronounced inputfrom orexin (hypocretin) containing cells as well as fromcocaine- and amphetamine-regulated transcript contain-ing (CART) neurons of the hypothalamus (Kirouac et al.,2006), and that both orexin and CART fibers synapse withPV cells projecting to the shell of ACC (Parsons et al.,2006). They proposed that PV links visceral/arousal sys-tems to limbic forebrain regions involved in behavioralresponses (Parsons et al., 2006).

Taken as a whole, the foregoing suggests that PV mayrepresent an important relay in the transfer of visceral/arousal, homeostatic, and circadian information to partsof the limbic system—thereby priming them (state ofreadiness) for behavioral responding. In this regard, PVneurons show elevated levels of c-fos expression duringperiods of wakefulness (compared to sleep) (Peng et al.,1995) as well as during stressful conditions (Chastrette etal., 1991; Bubser and Deutch, 1999; Sica et al., 2000;Otake et al., 2002)—which could be seen as heightenedstates of arousal. In view, then, of its pivotal role in limbiccircuitry, we sought to comprehensively examine the ef-ferent projections of PV in the rat.

Although the paratenial nucleus of the thalamus alsoappears to receive a vast array of afferent information(Chen and Su, 1990; Krout et al., 2002) and may selec-tively target structures of the limbic forebrain (Kelley andStinus, 1984; Carlsen and Heimer, 1986), little is knownregarding the connections of PT. The purpose, then, of thepresent study was to analyze, compare, and contrast ef-ferent projections of PV and PT nuclei of the midlinethalamus. We show that, with some important differences,the output of both PV and PT is mainly directed to themedial prefrontal and entorhinal cortices, the ventral sub-iculum of the hippocampus, claustrum, the dorsal and

ventral striatum, lateral septum, bed nucleus of stria ter-minalis, and to most of the amygdala, with a concentra-tion in the central and basal nuclei of the amygdala.

Materials and Methods

Single injections of Phaseolus vulgaris-leucoagglutinin(PHA-L) were made into either the PV or PT nuclei of themidline thalamus in 31 male Sprague–Dawley rats(Charles River, Wilmington, MA) weighing 275–400 g. Ofthe 31 injections, 10 were confined to PV, 8 were confinedto PT, 4 overlapped PV and PT, 4 overlapped PV and themediodorsal nucleus (MD); 3 overlapped PV and the in-termediodorsal nucleus (IMD), and 2 were localized to theinteranteromedial nucleus (IAM). Another 16 maleSprague–Dawley rats weighing 350–450 g received singleinjections of the retrograde fluorescent tracer FluoroGold(FG) (Fluorochrome, Denver, CO) into some PV and PTtargets: the central (CEA) and basolateral (BLA) nuclei ofthe amygdala and the core and shell of nucleus accum-bens. Of the 16 injections, seven were made in CEA orBLA of the amygdala and three were control injections inother nuclei of the amygdala. Of the seven injections in thecentral and basal nuclei of the amygdala, two were madein CEA, two in the basolateral nucleus (BLA), two in BLAand CEA, and one in BLA and the basomedial nucleus. Ofthe six injections in nucleus accumbens, three were local-ized to the core of ACC and three to the shell of ACC. Theexperiments were approved by the Florida Atlantic Uni-versity Institutional Animal Care and Use Committee andconform to all federal regulations and National Institutesof Health guidelines for the care and use of laboratoryanimals.

PHA-L procedures

Powdered lectin from PHA-L was reconstituted to 4–5%in 0.05 M sodium phosphate buffer (PB), pH 7.4. ThePHA-L solution was iontophoretically deposited in thebrains of anesthetized rats (sodium pentobarbital, 75 mg/kg, ip) by means of a glass micropipette with an outside tipdiameter of 40–60 �m. Positive direct current (5–10 �A)was applied through a Grass stimulator (Model 88) cou-pled with a high voltage stimulator (Frederick Haer, Bow-doinham, ME) at 2 seconds “on” / 2 seconds “off” intervalsfor 40–50 minutes. After a survival time of 7–10 days, ratswere deeply anesthetized with sodium pentobarbital andperfused transcardially with a heparinized buffered salinewash (100 mL/animal) followed by a fixative (4% parafor-maldehyde, 0.2–0.5% glutaraldehyde in 0.1 M phosphatebuffer, pH 7.4) (300–500 mL/animal). The brains wereremoved and stored for 2 days at 4°C in 30% sucrose in 0.1M PB. On the following day, 50-�m frozen sections werecollected in phosphate-buffered saline (PBS, 0.9% sodiumchloride in 0.01 M sodium phosphate buffer, pH 7.4) usinga sliding microtome. Six series of sections were taken. Acomplete series of sections was treated with 1% sodiumborohydride in 0.1 M PB for 30 minutes to remove excessreactive aldehydes. Sections were then rinsed in 0.1 M PB,followed by 0.1 M Tris-buffered saline (TBS), pH 7.6. Fol-lowing this, sections were incubated for 60 minutes atroom temperature (RT) in 0.5% bovine serum albumin(BSA) in TBS to minimize nonspecific labeling. The sec-tions were then incubated overnight at RT in diluent(0.1% BSA in TBS containing 0.25% Triton X-100) andbiotinylated goat anti PHA-L (Vector Labs, Burlingame,


214 R.P. VERTES AND W.B. HOOVER

CA) at a concentration 1:500. Sections were then washedin 0.1 M PB (4 � 8 minutes) and placed in a 1:500 con-centration of biotinylated rabbit antigoat immunoglobulin(IgG) and diluent for 2 hours. Sections were washed andthen incubated in a 1:100 concentration of peroxidase-avidin complex from the Elite kit (Vector) and diluent for1 hour. Following another 0.1 M PB wash the peroxidasereaction product was visualized by incubation in a solu-tion containing 0.022% 3,3� diaminobenzidine (DAB, Al-drich, Milwaukee, WI) and 0.003% H2O2 in TBS for 6minutes. Sections were then rinsed again in PBS (3 � 1minutes) and mounted onto chrome-alum gelatin-coatedslides. An adjacent series of sections from each rat wasstained with cresyl violet for anatomical reference. Sec-tions were examined using light and darkfield optics. In-jection sites, cells, and labeled fibers were plotted on rep-resentative schematic coronal sections through the brainusing sections adapted from the rat atlas of Swanson(1998). Brightfield and darkfield photomicrographs of in-jection sites and labeled fibers were taken with a NikonDXM1200 camera mounted on a Nikon Eclipse E600 mi-croscope. Digital images were captured and reconstructedusing Image-Pro Plus 4.5 (Media Cybernetics, SilverSprings, MD), and adjusted for brightness and contrastusing Adobe PhotoShop 7.0 (Mountain View, CA). Pat-terns of labeling were classified as light, moderate, anddense (Table 1), with ‘light’ referring to a few labeledfibers widely dispersed throughout a structure, ‘dense’ asa heavy concentration of labeled fibers generally occupy-ing a significant portion (or most) of a structure, and‘moderate’ between these two patterns.

FluoroGold procedures

FluoroGold (FG) (Fluorochrome) was dissolved in a 0.1M sodium acetate buffer (pH 4.0 to 5) to yield a 4–5%concentration. The FG solution was iontophoretically de-posited in the brains of anesthetized rats by means of aglass micropipette with an outside tip diameter of 25–50�m. Single FG injections were made into one of four struc-tures in separate rats: the central and basolateral nucleiof the amygdala and the core and shell of ACC. The pro-cedures for FG injections were basically the same as de-scribed for PHA-L injections, with the following excep-tions: 1) the outside tip diameter of the glassmicropipettes was 25–50 �m, and 2) the length of injec-tions was 2–10 minutes. Following a survival time of 7days, rats were deeply anesthetized with sodium pento-barbital and perfused transcardially with 100 mL of aheparinized saline wash followed by 450 mL of fixative(4% paraformaldehyde in 0.01 M sodium PB, pH 7.4). Thebrains were then removed and stored for 48 hours in asucrose solution (30% sucrose in 0.1 M PB) at 4°C. Follow-ing this, 50-�m coronal sections were taken on a freezingmicrotome and collected in 0.1 M PB and stored at 4°C. Sixseries of sections were taken. A complete series of sectionswas incubated in a sodium borohydride solution (1% so-dium borohydride in 0.1 M PB) for 30 minutes, andwashed with 0.1M PB four times at 6 minutes each (4 � 6min). The sections were then blocked in a Tris-salinesolution (0.5% BSA, Sigma Chemicals, St. Louis, MO;0.25% Triton X-100 in 0.1 M Tris-saline, pH 7.6) for 1hour. Following the blocking procedure the sections wereincubated for 48 hours at RT in a primary antiserumdirected against FG (rabbit anti-FluoroGold) (Chemicon,Temecula, CA) at a concentration of 1:1,000 in diluent.

Following incubation in the primary antiserum, sectionswere washed (4 � 6 min) in 0.1 M PB, then incubated in asecondary antiserum (biotinylated goat antirabbit IgG)(Vector) at a concentration of 1:500 in diluent for 2 hours.Sections were then washed again (4 � 6 minutes) and

TABLE 1. Relative Density of Anterogradely Labeled Fibers Produced byPHA-L Injections into PVa, PVp, and PT of the Midline Thalamus

Structure

Labeling

PVa PVp PT

TelencephalonCortex

Agranular insular, dorsal �� Agranular insular, posterior � �� Agranular insular, ventral � � �Agranular, lateral (primary motor) � � �Agranular, medial (secondary motor) � � �Anterior cingulate, dorsal � � ��Anterior cingulate, ventral � � ��Ectorhinal � �� Entorhinal � �� Frontal polar, lateral � � �Frontal polar, medial � � ��Granular insular � �� Infralimbic � �� Lateral orbital � � �Medial orbital � � ��Parietal � � �Perirhinal � �� Piriform � ��Prelimbic �� Retrosplenial � � �Subiculum, ventral �� Somatosensory, secondary � � �Ventral Orbital �� Ventrolateral Orbital � � �

Accumbens n.Core �� Shell ��

AmygdalaAnterior amygdaloid area � � ��Basolateral n. � �� Basomedial n. �� Central n. �� Cortical n. � �� Lateral n. � �� Medial n. � �� Posterior n. � � ��Amygdaloid-piriform area � � ��

Bed n. of stria terminalis �� Claustrum �� Diagonal band of Broca, horizontal � � �Endopiriform n. � � �Globus pallidus � � �Medial preoptic area � �� Olfactory tubrical �� Septum

Medial � � �Lateral � ��

Striatum �� Substantia innominata � � �Tenia tecta � ��

DiencephalonHypothalamus

Arcuate n. �� Dorsomedial n. � �� Lateral hypothalamic area � � ��Paraventricular n. � � �Posterior hypothalamus � � �Suprachiasmatic n. �� Supramammillary n. � � �

ThalamusCentral medial n. � � �Interanteromedial n. � � �Mediodorsal n. � � �Parafascicular n. � � �Parataenial n. � � �Paraventricular n. � � �Reticular n. � � �Reuniens n. � � �Rhomboid n. � � �

�, light labeling; ��, moderate labeling; ��, dense labeling; �, absence of labeling;n, nucleus



Fig. 1. High-power brightfield photomicrographs at two levels ofmagnification of PHA-L injections sites in the anterior paraventricu-lar nucleus (A,B), the posterior paraventricular nucleus (C,D), andthe paratenial nucleus (E,F) of the dorsal midline thalamus. Note

clearly visible PHA-L filled cells in PVa (B), PVp (D), and PT (F). Scalebar � 375 �m for A,E; 200 �m for B; 400 �m for C; 300 �m for D; 225�m for F.



incubated in avidin-biotin complex (Vector) at a 1:100concentration in diluent for 1 hour. After a final set of 4 �6 minute rinses the peroxidase reaction product was visu-alized by incubation in a solution containing 0.022% ofDAB (Aldrich), 0.015% nickel chloride (NiCl), and 0.003%

H2O2 in TBS for 6 minutes. Sections were rinsed again inPBS (3 � 1 minutes) and mounted onto chrome-alumgelatin-coated slides. An adjacent series of representativesections from each rat was stained with cresyl violet foranatomical reference. The resulting material was pro-

Fig. 2. Schematic representation of labeling present in select sections through the forebrain andmidbrain (A–N) produced by a PHA-L injection (dots in I,J) in the anterior part of the paraventricularnucleus of the thalamus (case 6). Sections modified from the rat atlas of Swanson (1998). See list forabbreviations.



cessed for presentation as described for the PHA-L sec-tions.

Results

The pattern of distribution of projections from the PVand PT nuclei of the thalamus are described. Figure 1shows sites of injection in the anterior PV (PVa) (Fig.1A,B), the posterior PV (PVp) (Fig. 1C,D), and PT (Fig.1E,F) at two levels of magnification. As depicted, PHA-L-filled cells are restricted to respective nuclei. The patternsof labeling obtained with the schematically depicted casesare representative of patterns seen with nonillustratedcases.

Anterior paraventricular nucleus (PVa)(case 6)

Figure 2 schematically depicts the pattern of distribu-tion of labeled fibers following a PHA-L injection in theanterior part of PV (case 6). As shown, labeled fiberscoursed ventrolaterally from the site of injection (Fig. 2F)within the thalamic peduncle to regions of the lateralhypothalamus and from there took three principal routes.A major contingent continued ventrolaterally in amyg-dalopetal pathways to reach the amygdala and surround-ing regions of cortex, others coursed rostrally to the ante-rior forebrain primarily bound for the ventral striatumand the prefrontal cortex or caudally en route to regions of

Figure 2 (Continued)



the hypothalamus. Some labeled fibers of the ascendingbundle joined the stria terminalis and traveled with it toreach the amygdala and adjacent regions of cortex.

Overall, labeling was light at the anterior pole of theforebrain (Fig. 2A,B). A small collection of labeled fiberswas seen along the medial wall of the prefrontal cortex(PFC), mainly within the anterior prelimbic (PL) cortex.Some diffuse labeling was also observed in the dorsaltenia tecta (TTd) (Fig. 2A,B).

Further caudally at the rostral forebrain (Fig. 2C,D),labeling was primarily confined to PL of the mPFC and torostral aspects of nucleus accumbens (ACC). As depictedin Figure 3A, labeled fibers mainly encircled the outerboundaries of ACC, and were much less concentrated inthe core of the rostral ACC. Additional labeled sites werethe anterior claustrum (CLA), ventromedial regions of thedorsal striatum bordering ACC (Fig. 2D), and to a lesserdegree the dorsal agranular insular cortex (AId) (Fig. 2C).There was a noticeable lack of labeling at this level (Fig.2C), as well as caudally, over most of the cortical mantle.Labeling was stronger on the left than on the right side ofthe brain (Fig. 2A–M), reflecting the fact that the PVinjection was slightly lateralized to the left side (Fig.1A,B).

At septal levels (Fig. 2D–H), labeling was pronouncedand relatively restricted to the core and shell regions ofACC, to ventral aspects of the lateral septum (LS) (Figs.2F, 3B), and to the bed nucleus of the stria terminalis(BST). The dense labeling within the shell of ACC and toa slightly lesser degree in the core of ACC is depicted inthe photomicrographs of Figure 3B, while equally pro-nounced labeling of BST, above and below the anteriorcommissure, is shown in Figure 3C. Outside of these sites,CLA, the olfactory tubercle (OT), and ventral regions ofthe dorsal striatum were moderately labeled, while theventral globus pallidus (Fig. 2H) was lightly labeled.

At mid levels of the forebrain (Fig. 2I–K), labeling wasmainly confined to the amygdala and parts of the hypo-thalamus. The arcuate and suprachiasmatic (SCN) (Fig.2I) nuclei of the hypothalamus were moderately labeled.Within the (rostral) amygdala, labeling was very heavywithin the central nucleus (CEA) (Fig. 2J,K), particularlywithin the lateral CEA (Fig. 3D), moderately dense in thebasomedial (BMA) and basolateral (BLA) nuclei, and rel-atively light in the medial and cortical nuclei of amygdala.Some labeled fibers were also present on the lateral con-vexity of cortex within the posterior agranular insularcortex, rostrally (Fig. 2I,J) and in the perirhinal (PRC),rostral entorhinal (EC) and piriform cortices, caudally(Fig. 2K).

At further caudal levels of the forebrain (Fig. 2L,M) andthe rostral midbrain (Fig. 2N), the bulk of labeled fiberswas localized to the amygdala and surrounding regionsincluding caudal parts of the dorsal striatum (Fig. 2L,M),deep layers of the perirhinal, entorhinal and piriform cor-tices (Fig. 2L–N), and the ventral subiculum of the hip-pocampus (Fig. 2N). Within the amygdala, labeling waspredominantly restricted to the basal nuclei—densewithin BMA and moderate within the medial part of BLA.Some labeled fibers were also present in the posterior PV(PVp) and in the dorsomedial nucleus of the hypothala-mus (Fig. 2L,M).

Posterior paraventricular nucleus (PVp)(case 32)

Anterior and posterior PV injections largely gave rise tosimilar patterns of labeling but, as described below, over-all density of labeling was stronger with PVp than withPVa injections.

Labeled fibers from PVp mainly coursed rostrallythrough the dorsal thalamus (Fig. 4K–N) and approxi-mately at the level of the rostral pole of the hippocampus(Fig. 4I,J) turned ventrolaterally to exit the thalamus.From there, they either continued on the same trajectoryto reach the amygdala and surrounding regions of thedorsal striatum and cortex or ascended or descendedthrough the medial forebrain bundle (MFB) en route tothe basal forebrain and prefrontal cortices, rostrally, or toparts of the hypothalamus, caudally.

Similar to PVa, labeling at the anterior pole of theforebrain (Fig. 4A,B) was generally moderate and mainlypresent in inner layers of the anterior PL and anteriorcingulate (AC) cortices and to considerably lesser degreesin medial frontal polar (FPm), medial orbital (MO), anddorsal agranular insular (AId) cortices. Moderate num-bers of labeled axons were also visible in TTd.

Further caudally at the rostral forebrain (Fig. 4C), la-beled fibers spread widely over ventral aspects of the brainmainly localized to the ventral mPFC, claustrum, dorsalagranular insular cortex (AId), rostral ACC, and the ol-factory tubercle (OT). As depicted (Fig. 4C), labeling wasquite dense in the inner layers of the infralimbic (IL) andprelimbic cortices and somewhat less pronounced in AId,CLA, rostral ACC, and OT. A few labeled fibers were alsoseen in AC.

The main targets of labeled fibers further caudally inthe forebrain were the dorsal and ventral striatum (Fig.4D–F). As depicted schematically (Fig. 4D,E) and in themicrograph of Figure 5A, the shell of ACC (ACCs) wasintensely labeled. With the exception of the region sur-rounding the anterior commissure, which was heavily la-beled, the core of ACC (ACCc) was moderately labeled.Unlike PVa, significant numbers of labeled axons werealso present in ventral aspects of the dorsal striatum (CP),progressively thinning from the ventrolateral to dorsome-dial CP. Other moderately to heavily labeled sites at theselevels were ventral LS, OT, CLA, and AId (Fig. 4D–F).

Immediately caudal to ACC (Fig. 4F–H), labeled axonsspread densely throughout the extent of the bed nucleus ofthe stria terminalis (BST) and were also present in size-able numbers in medial aspects of CP, CLA, OT, AId, andthe suprachiasmatic nucleus (SCN). Figure 5B shows adiscrete group of labeled axons bilaterally within SCN.Additional light to moderately labeled sites included theposterior agranular insular cortex (AIp) (with some exten-sion dorsally into the granular insular cortex), substantiainnominata (SI), medial preoptic area (MPO) and the glo-bus pallidus (GP) (Fig. 4F–H).

At mid to caudal levels of the forebrain (Fig. 4I–N),labeled fibers were mainly confined to the dorsal stria-tum and to the amygdala, spreading massively through-out the amygdala. As shown (Figs. 4I–N, 6A–D), labeledfibers virtually blanketed the amygdala, with the dens-est concentration in the central (CEA) (Fig. 6A,B) andbasomedial (BMA) (Fig. 6A–D) nuclei of amygdala. Themedial (MEA) and basolateral nuclei of amygdala werealso fairly heavily labeled, whereas the lateral and



parts of the anterior and posterior cortical nuclei ofamygdala were moderately labeled (Fig. 4I–N). At preand beginning levels of the hippocampus (Fig. 4H–K), arelatively narrow band of labeled fibers within the me-

dial CP, abutting the globus pallidus, was observed.Labeling was densest ventrally in medial CP (Fig.4I–M) at its point of merger with medial aspects of theamygdala.

Fig. 3. A–D: Low-magnification darkfield photomicrographs oftransverse sections through anterior (A–C) and posterior (D) regionsof the forebrain depicting patterns of labeling in rostral and caudalnucleus accumbens (ACC) (A,B), the bed nucleus of the stria termi-nalis (C), and the amygdala (D) produced by a PHA-L injection intoanterior paraventricular nucleus of thalamus. A: Note that labeledfibers mainly encircle but largely avoid the central core of the rostralACC, and also note significant labeling in the prelimbic cortex (PL) ofthe medial prefrontal cortex. B: By contrast with the rostral ACC (A),

labeled fibers distribute massively to caudal part of ACC. Note pro-nounced labeling in both the shell and core of ACC, with additionallabeling in the adjacent lateral septum (LS) and ventromedial parts ofthe dorsal striatum (caudate-putamen) (CP). C: Note strong terminallabeling in BST above and below the anterior commissure. D: Note adense aggregate of labeled fibers in the central medial and medialpart of the medial part of the basomedial nucleus and prominent butless dense labeling in the basolateral nucleus of the amygdala. Scalebar � 550 �m for A; 500 �m for B–D. See list for abbreviations.



Further caudally (Fig. 4L–N), labeled fibers were foundto extend laterally from the medial CP to occupy most ofthe mediolateral expanse of the caudal CP. In addition, asrostrally, posterior parts of the lateral, central, basal

(BMA and BLA), and cortical nuclei of amygdala weremoderately to densely labeled (Fig. 4L–N). Significantnumbers of labeled axons were also visible within innerlayers of the parahippocampal and piriform cortices; that

Fig. 4. Schematic representation of labeling present in select sections through the forebrain andmidbrain (A–O) produced by a PHA-L injection (dots in N) in the posterior part of the paraventricularnucleus of the thalamus (case 32). Sections modified from the rat atlas of Swanson (1998). See list forabbreviations.



is, a continuous column in layers 5 and 6 stretching fromthe ectorhinal, perirhinal, and entorhinal cortices to thepiriform cortex (Fig. 4L–N). Although most regions of thediencephalon were devoid of labeled fibers (Fig. 4L–N),

moderate numbers were present in the dorsomedial nu-cleus of the hypothalamus (DMH) (Fig. 5C) and a fewwithin the midline thalamus–reuniens (RE) and rhomboid(RH) nuclei.

At the level of the midbrain (Fig. 4O) moderate numbersof labeled fibers were present in the caudal perirhinalcortex, lateral EC, and the ventral subiculum of the ven-tral hippocampus. Although labeling progressivelythinned caudally, labeled axons continued to be present inlateral EC and the ventral subiculum throughout caudalreaches of the brain (not shown). With a few exceptions,there was a general absence of labeling at the rostralmidbrain (Fig. 4O) and throughout the brainstem.

Paratenial nucleus (PT) (case 27)

As depicted (Fig 1E,F), the injection in the paratenialnucleus (PT) was confined to left PT, and accordinglylabeling was virtually restricted to the left side of thebrain. Labeling was minimal contralateral to the injec-tion. Similar to PV, labeled fibers from PT exited ventro-laterally through the thalamus and then either continuedon the same course to the amygdala or ascended throughthe MFB to the anterior forebrain or descended in theMFB to parts of the caudal diencephalon.

At anterior pole of the forebrain (Fig. 7A,B), virtuallythe entire medial wall of the mPFC was densely labeled.This includes the medial frontal polar, prelimbic (PL), andmedial orbital (MO) cortices, rostrally (Fig. 7A) and theanterior cingulate, PL, and MO cortices, further caudally(Fig. 7B). This is shown in the photomicrographs of Figure8A,B. As depicted, labeled fibers spread to all layers ofrespective cortices, but were most heavily concentrated inlayers 2/3 of PL and the ventrally adjacent MO. In addi-tion, moderate numbers of labeled fibers were present inthe ventral orbital cortex (VO) and TTd, but considerablyfewer in AId.

Further caudally in the anterior forebrain (Fig. 7C),labeling remained pronounced along the medial wall ofthe ventral mPFC, particularly pronounced in layers 1/3 ofthe infralimbic (IL) and prelimbic cortices (Figs. 7C, 8C,9). Equally dense labeling was observed within the rostralACC and parts of OT. Additional lightly to moderatelylabeled sites included AId, AC, and the CLA. This patternof labeling is depicted in the brightfield photomicrographof Figure 9.

At early septal levels (Fig. 7D–F), labeled axons wereprimarily localized to the ventral mPFC, CP, ventral stri-atum, CLA, and AId. Labeling was pronounced (or mas-sive) in IL and PL of the mPFC, ventromedial sectors ofCP, the core and shell of ACC, and parts of OT (Fig. 7D–F).Some regions of AC, AId, and LS were also heavily labeled(Fig. 7E,F). As depicted schematically (Fig. 7D–F) and in

Fig. 5. A–C: Low-magnification darkfield photomicrographs oftransverse sections through the forebrain depicting patterns of label-ing produced by a PHA-L injection into posterior paraventricularnucleus. A: Note the massive labeling throughout the shell division ofthe nucleus accumbens (ACCs) and intense but lesser labeling in thecore of ACC (ACCc) surrounding the anterior commissure. B: Note thestrong labeling bilaterally in the suprachiasmatic nucleus of the hy-pothalamus (SCN) above the optic chiasm. C: Note the pronouncedlabeling bilaterally in the dorsomedial nucleus (DMh) of the hypothal-amus lateral to the third ventricle. Scale bar � 500 �m for A; 250 �mfor B; 300 �m for C. See list for abbreviations.



the photomicrograph of Figure 10A, labeling was unevenmediolaterally across ACC and within bordering regionsof CP; that is, dense in the internal (medial) shell of ACC,considerably lighter in the lateral shell (with some rela-tively clear pockets), very strong in the (dorsal) core ofACC, extending into the ventromedial CP, and moderatein the lateral CP.

At caudal levels of the septum (Fig. 7G,H) significantnumbers of labeled axons were present in rostral BST, onthe lateral and ventral border of the anterior commissure,and medially within CP abutting lateral wall of the lateralventricle, dorsal to BST. Light to moderate labeling wasalso observed in AC, LS, ventromedial CP, olfactory tu-bercle, and CLA.

There was marked decline in labeling further caudallyin the forebrain (Fig. 7I–K) with labeled fibers mainly

observed in the rostral amygdala and adjoining regions ofthe piriform cortex. As shown (Fig. 7I–K), labeled fibersspread widely (and moderately) throughout the amygdalato the anterior amygdaloid area (AA), CEA, MEA, BLA,BMA, and the anterior cortical nucleus (COAa). There wasa noticeable absence of labeling in the core of CEA (Fig7J,K). Other lightly to moderately labeled sites were AC,dorsomedial CP, CLA, and anterior and lateral nuclei ofthe hypothalamus.

At mid-levels of the forebrain (Fig. 7L–N), labeledfibers were essentially confined to the ventrolateral sec-tor of the brain; that is, to CP, to the amygdala, and tothe perirhinal, entorhinal, and piriform cortices. Anintensely labeled band of tissue stretching diagonallythrough the amygdala was observed (Fig. 7L–N, 10B)that included medial aspects of the lateral and basolat-

Fig. 6. A–D: Series of low-magnification darkfield photomicro-graphs of transverse sections rostrocaudally through the forebrain(A–D) depicting patterns of labeling within the amygdala produced bya PHA-L injection into posterior paraventricular nucleus. A,B: Notevery intense dense labeling in the central (CEA), basomedial (BMA)

and basolateral nuclei of amygdala, and prominent but less denselabeling in the parts of the medial (MEA), lateral (LA), and anteriorcortical nuclei of amygdala. C,D: Note labeling at caudal levels of theamygdala mainly confined to BMA and BLA. Scale bar � 750 �m. Seelist for abbreviations.



eral amygdala and virtually the extent of posteriorBMA. As rostrally, the core of CEA was largely devoid oflabeled fibers (Fig. 7L), but moderate numbers wereseen in the capsular CEA as well as in the posterior,

amygdaloid-piriform area, and posterior cortical nucleiof the amygdala (Fig. 7L–N). A few labeled fibers werealso present in RE, the zona incerta (ZI), and through-out the lateral hypothalamus.

Fig. 7. Schematic representation of labeling present in select sections through the forebrain andmidbrain (A–O) produced by a PHA-L injection (dots in I,J) in the paratenial nucleus of the thalamus(case 27). Sections modified from the rat atlas of Swanson (1998). See list for abbreviations.



At the rostral midbrain (Fig. 7O), labeled fibers con-tinued to mainly occupy ventrolateral regions of thebrain localized to perirhinal cortex, lateral EC, and theventral subiculum of the hippocampus. This pattern oflabeling is depicted in the micrographs of Figure 11A–Cshowing prominent label ing in these areas—particularly dense rostrocaudally throughout the lat-eral EC. The few labeled fibers present dorsally in theretrosplenial cortex (Fig. 7O) mainly appeared boundfor the dorsal subiculum which was lightly labeled(Fig. 7O).

Retrograde tracing experiments

Two major destinations of labeled fibers of PVa, PVp,and PT were the nucleus accumbens (shell and core) andthe amygdala—mainly CEA and basal nuclei (see above).To confirm anterograde findings and provide further in-formation on the distribution of PV and PT fibers to thesesites, retrograde injections (FG) were made in ACC andthe amygdala and patterns of labeling in PV and PTdetermined.

Figure 12 shows FG injections in the shell (Fig. 12A)and the core (Fig. 12D) of ACC together with patterns ofcell labeling in PVa and PT (Fig. 12B,E) and PVp (Fig.12C,F) obtained with these injections. As depicted: 1) la-beled cells were present in PVa, PVp, and densely in PTwith injections in the shell (ACCs) and core (ACCc) ofACC; 2) labeling was heavier in PVa with ACCc than

ACCs injections; and 3) labeling was slightly stronger inPVp with ACCc than ACCs injections. This supports an-terograde results showing pronounced terminal labelingin the shell and core of ACC with PVa, PVp, and PTinjections.

Figure 13 shows FG injections in the rostral BLA (Fig.13A) and rostral CEA (Fig. 13D), together with patternsof cell labeling in PVa and PT (Fig. 13B,E) and PVp (Fig.13C,F) produced by these injections. As depicted, thereis a small number of labeled neurons in PT (Fig. 13B)with the BLA injection and fewer still in PT (Fig. 13E)with the CEA injection. This is consistent with antero-grade findings showing light terminal labeling in rostralBLA (Fig. 7I,J) and general lack of labeling in rostralCEA (Fig. 7I,J) with PT injections. FG injections in BLAgave rise to pronounced cell labeling in PVp (Fig. 13C), butlight labeling in PVa (Fig. 13B), while those in CEA pro-duced significant labeling in both PVa (Fig. 13E) and PVp(Fig. 13F). This is consistent with anterograde results dem-onstrating strong terminal labeling in rostral CEA with PVa(Fig. 2I,J) and with PVp injections (Figs. 4I,J, 6A,B), as wellas weak labeling in BLA with PVa injections (Fig. 2I,J) anddense labeling in BLA (Figs. 4I,J, 6A) with PVp injections.As depicted, labeled cells are also present in RE (Fig. 13B,E),the intermediodorsal (IMD), and the central medial (CM)nuclei of the thalamus (Fig. 13C,F) with BLA and CEAinjections.

Fig. 8. A–C: Series of rostrocaudally (A–C) aligned low-magnification darkfield photomicrographs of transverse sectionsthrough the anterior forebrain depicting patterns of labeling withinthe medial prefrontal cortex (mPFC) produced by a PHA-L injectioninto the paratenial nucleus of the thalamus. Note the presence of

intense labeling along the ventral medial wall of the mPFC mainlyconfined to the prelimbic (PL) (A–C), medial orbital (MO) (A,B), andinfralimbic (C) (IL) cortices. As depicted, labeling was particularlydense in layers 1 and 3 of these prefrontal fields. Scale bar � 750 �m.



Discussion

We examined, compared, and contrasted the efferentprojections of the PV and PT nuclei of the dorsal midlinethalamus in the rat. The main (or virtually sole) targets ofPV and PT were ‘limbic/limbic related’ structures of theforebrain. With the possible exception of the piriform cor-tex, there was essentially lack of PV/PT projections to‘nonlimbic’ regions of the cortex including sensorimotor,special sensory, or associational cortices as well as fewprojections to most of the thalamus and hypothalamus. Asdeveloped below, based on widespread afferents from thebrainstem and hypothalamus coupled with output to se-lect structures of the limbic forebrain, PV/PT appear crit-ical for routing visceral/emotional information to struc-tures of the limbic forebrain, including the limbic cortex,in the control of goal-directed behaviors.

Overview of PV projections and comparisonsbetween PVa and PVp projections

The main targets of PV (anterior and posterior parts)were the prelimbic (PL), dorsal agranular insular, perirhi-nal (PRC) and entorhinal cortices, the ventral subiculumof the hippocampus, the claustrum, the lateral septum

(LS), the core and shell of ACC, OT, BST, several nuclei ofthe amygdala, including the lateral, medial, central, basal(BLA and BMA), and the anterior and posterior corticalnuclei, and the suprachiasmatic (SCN), arcuate, and dor-somedial nuclei of the hypothalamus. Secondary targetswere the anterior cingulate and ectorhinal cortices, dorsaltenia tecta (TTd), the medial preoptic area, reuniens (RE),and rhomboid nuclei of the thalamus and the lateral hy-pothalamus.

There is a significant overlap in projections from theanterior (PVa) and posterior (PVp) PV (Figs. 2, 4; Table 1).With some exceptions, PVp is the source of stronger pro-jections to most commonly innervated sites. Perhaps themost significant difference between PVa and PVp projec-tions is that PVa distributes minimally to the dorsal stri-atum (caudate-putamen), whereas PVp projects quitemassively to CP, mainly to medial/ventromedial regions ofCP. In addition, while PVa and PVp project commonly tothe amygdala, PVp distributes more widely and heavilythroughout the amygdala than PVa, particularly to thebasal nuclei of amygdala. On the other hand, PVa is thesource stronger projections to the ventral subiculum of thehippocampus.

Overview of PT projections andcomparisons with PV projections

The main targets of PT were the medial frontal polar(FPm), anterior cingulate, prelimbic, infralimbic, medialorbital, dorsal agranular insular, piriform and entorhinalcortices, the ventral subiculum of hippocampus, the claus-trum, the core and shell of nucleus accumbens, the medialstriatum (CP), BST, and caudal parts of the central andbasal nuclei of amygdala. PT also distributes to the ven-tral orbital and perirhinal cortices, the dorsal subiculumof hippocampus, lateral septum, olfactory tubercle, medialand cortical nuclei of amygdala, RE of thalamus, and thelateral hypothalamus.

Although there is considerable overlap in PT and PVprojections, there are several important differences be-tween the two sets of projections. PT sends considerablystronger projections than PV to the mPFC, to the lateralentorhinal cortex, to the ventral subiculum, and to ante-rior regions of the dorsal and ventral striatum. Differencesare particularly notable with respect to the mPFC. PTstrongly targets the mPFC, distributing throughout theventral mPFC to the medial frontal polar, medial orbital(MO), anterior cingulate, prelimbic and infralimbic corti-ces, and particularly heavily in outer layers (1 and 3) ofMO, PL, and IL (Fig. 8A–C). By contrast, the projections ofPV (PVa and PVp) to the mPFC are modest and mainlyconfined to IL and PL. With respect to nucleus accumbens(ACC), PT distributes more heavily to the rostral pole (Fig.9) and core of ACC (Figs. 10A, 12E), but less densely to theshell of ACC (Figs. 3B, 5A) than does PV. Unlike PVa, butsimilar to PVp, PT strongly targets the dorsal striatum.PT fibers terminate densely (and selectively) in the ros-tromedial CP, dorsal to the core of ACC (Fig. 10A), whilePVp distributes rostrocaudally throughout CP and heavilyto the caudal CP—a region basically devoid of fibers fromPT. Finally, in contrast to robust PV projections to virtu-ally the entire amygdala, PT distributes significantly tocaudal, but at best modestly, to rostral parts of the amyg-dala (see Fig. 13B,E).

Fig. 9. Low-magnification brightfield photomicrograph of a trans-verse section through the anterior forebrain depicting patterns oflabeling within the anterior cingulate (AC), prelimbic (PL), infralim-bic (IL), and dorsal agranular insular cortices, the olfactory tubercle(OT), and the rostral pole of the nucleus accumbens (ACC) producedby a PHA-L injection in the paratenial nucleus of thalamus. Scalebar � 500 �m. See list for abbreviations.



Fig. 10. A,B: Low-magnification darkfield photomicrographs oftransverse sections through the forebrain depicting patterns of label-ing the dorsal and ventral striatum (A) and the amygdala (B) pro-duced by a PHA-L injection into the paratenial nucleus of the thala-mus. A: Note the dense labeling, but uneven labeling (sparsely labeledpockets) in the shell of nucleus accumbens (ACC) and the massivelabeling in the core of ACC (dorsal/dorsomedial to the anterior com-

missure) with a continuation of equally dense labeling into ventrome-dial parts of the dorsal striatum (caudate putamen, CP). B: Noteheavy labeling in parts of the lateral and basolateral nuclei, some-what less pronounced labeling in the basomedial nucleus and anabsence of labeling in the lateral part of the central nucleus of amyg-dala. Scale bar � 550 �m for A; 500 �m for B. See list for abbrevia-tions.



PV projections: comparisons with previousstudies

As discussed, the output of PV is restricted; that is, PVprojects to a limited number of sites, but quite massivelyto them. The foremost PV targets are nucleus accumbens,bed nucleus of stria terminalis, and the amygdala.

The most complete analysis of PV projections was anearly report by Moga et al. (1995). Our findings werecomparable to theirs for the anterior PV (PVa) but differed

considerably for the posterior PV. In accord with Moga etal. (1995), we demonstrated pronounced PVa projectionsto the shell of ACC and to the central and basomedialnuclei of amygdala, but unlike them, also described sub-stantial PVa projections to the core of ACC as well as tothe medial, basolateral, and cortical nuclei of amygdala.On the other hand, they demonstrated denser projectionsto nuclei of the hypothalamus including the retrochias-matic nucleus, subparaventricular zone, and the ventro-medial nucleus of the hypothalamus.

With respect to PVp, however, Moga et al. (1995) re-ported that PVp projections were much lighter overallthan PVa projections, while we generally found the oppo-site: stronger PVp than PVa projections to most sites.Further, Moga et al. (1995) described an essential lack ofPVp projections to several sites in which we observedthem including the infralimbic, piriform, perirhinal andagranular insular cortices, the ventral subiculum, medialregions of the striatum, posterior BLA and BMA, and mostof the hypothalamus. The reasons for these differences areunclear but could involve differences in size and locationsof the PVp injections.

PV projections to emotional/visceral associated fore-

brain areas. PV distributes to several forebrain sitesassociated with emotional behavior including the in-fralimbic cortex, the lateral septum, bed nucleus of striaterminalis, and almost the entire amygdala—with mas-sive projections to CEA.

In accord with present findings, previous reports, usingvarious tracers, have described significant PV projectionsto the infralimbic cortex in rats (Berendse and Groenewe-gen, 1991; Conde et al., 1995; Moga et al., 1995; Bubserand Deutch, 1998; Otake and Nakamura, 1998; Pinto etal., 2003), mainly targeting inner layers (5/6) of IL (Be-rendse and Groenewegen, 1991; Pinto et al., 2003). Werecently identified labeled cells rostrocaudally throughoutPV following retrograde tracer injections in IL (Hooverand Vertes, 2007). PV also distributes substantially toarea 25 (or the infralimbic cortex) in primates (Hsu andPrice, 2007). Moga et al. (1995) reported that PVa projectsdensely, whereas PVp sparsely (or not at all) to the lateralseptum (LS). We showed that both PVa and PVp project toLS, but similar to Moga et al. (1995) found that the majoroutput was from PVa. Consistent with this, Risold andSwanson (1997) described labeled cells throughout PVfollowing FG injections in LS, but progressively fewer cellsat successive caudal levels of PV.

Few reports have examined PV projections to the bednucleus of stria terminalis (Moga et al., 1995; Van derWerf et al., 2002). In general accord with present findings,Moga et al. (1995) reported that PV distributes heavily torostral and lateral parts (subnuclei) of BST. Although theefferent projections of BST have been fairly extensivelyexamined (Dong et al., 2001; Gu et al., 2003; Dong andSwanson, 2004, 2006), to our knowledge, only a singleearly report by Weller and Smith (1982) examined affer-ents to BST. They showed PV and PT are virtually the solesources of thalamic input to BST, distributing signifi-cantly to BST.

We showed that PV distributes massively throughoutthe amygdala, and with the exception of parts of thecaudal amygdala, to most subnuclei of the amygdala. Theforemost PV targets are the central and basal nuclei of theamygdala. In accord with present findings, an early exam-ination of thalamic afferents to the amygdala (Ottersen

Fig. 11. A–C: Series of rostrocaudally aligned low magnificationdarkfield photomicrographs of transverse sections through the fore-brain depicting patterns of labeling within the ventral subiculum(SUBv) and lateral entorhinal cortex (ECl) produced by a PHA-Linjection into the paratenial nucleus of the thalamus. Note stronglabeling in ECl as well as in the molecular layer of SUBv. Scale bar �500 �m. See list for abbreviations.



Fig. 12. A–C: Series of low-magnification brightfield photomicro-graphs of transverse sections through the forebrain depicting the siteof a FluoroGold injection in the shell of nucleus accumbens (ACCs) (A)and patterns of retrogradely labeled cells within the anterior para-ventricular (PV) and paratenial (PT) nuclei (B) and the posteriorparaventricular nucleus (C) produced by this injection. Note signifi-cant numbers of retrogradely labeled neurons in PT, moderate num-bers in posterior PV, and relatively few in the anterior PV with thisinjection. D–F: Series of low- magnification brightfield photomicro-

graphs of transverse sections through the forebrain depicting the siteof a FluoroGold injection in the core of nucleus accumbens (ACCs) (D)and patterns of retrogradely labeled cells within the anterior para-ventricular (PV) and paratenial (PT) nuclei (E) and posterior para-ventricular nucleus (F) produced by this injection. Note significantnumber of retrogradely labeled neurons in anterior PV and PT andmoderately number in posterior PV produced by this injection. Scalebar � 500 �m for A,D; 350 �m for B,E; 400 �m for C,F. See list forabbreviations.



and Ben-Ari, 1979) described widespread PV (and PT)projections to the amygdala, stating that “the paraven-tricular and paratenial nuclei of the thalamus were foundto project throughout the amygdaloid complex.” Severalsubsequent studies have confirmed pronounced PV projec-tions to CEA, BMA, and BLA (Berendse and Groenewe-gen, 1990; Su and Bentivoglio, 1990; Turner and Herken-ham, 1991; Moga et al., 1995; Peng and Bentivoglio, 2004).

PV projections to ‘cognitive-associated’ forebrain

areas. Groenewegen and colleagues (Room et al., 1985;Groenewegen et al., 1990) initially defined a system ofconnections (or loop) from the prelimbic cortex � ventralstriatum � ventral pallidum � MD of thalamus � PLwhich they termed the ‘PL circuit.’ The ‘PL circuit’ hassubsequently been expanded to include several additionalstructures; principal among them are the dorsal agranu-lar insular cortex (AId), hippocampus/parahippocampus,the basolateral amygdala, parts of midline thalamus andthe ventral tegmental area (VTA) (Vertes, 2006). PL andits interconnected circuitry serve a recognized role in cog-nitive functions (Laroche et al., 2000; Groenewegen andUylings, 2000; Vertes, 2006). PV distributes to severalstructures of prelimbic circuit: PL, AId, ACC, EC, theventral subiculum, BLA, and VTA.

We showed that PV (PVa and PVp) distributes: 1) se-lectively to IL and PL of the ventral mPFC; 2) moreheavily to PL than to IL; and 3) rostrocaudally throughoutPL, terminating most densely in inner layers of PL. Thesefindings are consistent with previous descriptions of sig-nificant PV projections to PL (Berendse and Groenewegen,1991; Conde et al., 1995; Moga et al., 1995; Bubser andDeutch, 1998; Otake and Nakamura, 1998; Pinto et al.,2003; Hoover and Vertes, 2007).

We found that PVa and PVp distribute massivelythroughout the shell and core of ACC. Although earlyreports showed that PV (and PT) strongly target ACC(Groenewegen et al., 1980; Newman and Winans, 1980;Beckstead, 1984; Jayaraman, 1985; Phillipson and Grif-fiths, 1985), Groenewegen and colleagues (Berendse et al.,1988; Berendse and Groenewegen, 1990) were the first toshow that each of the midline nuclei distribute to select,and only partially overlapping, territories of the ventralstriatum. Regarding PV, they reported that of the midlinenuclei of thalamus, PV was the predominant source ofafferents to the shell of ACC, and while also pronounced tothe core, were shared by other midline thalamic groups tothe core (Berendse and Groenewegen, 1990). Several sub-sequent studies have confirmed ‘massive’ PV projectionsto ACC, and further showed that a fairly significant per-centage of PV fibers to ACC collateralize to other sites(Meredith and Wouterlood, 1990; Su and Bentivoglio,1990; Brog et al., 1993; Freedman and Cassell, 1994; Mogaet al., 1995; Bubser and Deutch, 1998; Otake and Naka-mura, 1998; Erro et al., 2002; Pinto et al., 2003; Parsons etal., 2006, 2007), mainly to the mPFC (Bubser and Deutch,1998; Otake and Nakamura, 1998) and to the amygdala(Su and Bentivoglio, 1990).

Finally, in accord with previous reports (Berendse et al.,1988; Berendse and Groenewegen, 1990), we found thatPV fibers distribute in a nonhomogeneous (or ‘patch/matrix’) manner to the nucleus accumbens; that is, re-gions of dense innervation interspersed with relativelyfiber free zones (Figs. 2, 3B, 4, 5A). Berendse et al. (1988)reported that densely PV-innervated regions of the rostralACC and sparsely innervated areas of the caudomedial

ACC overlap with zones of strong and weak enkephalinimmunoreactivity, respectively. We did not immunostainfor enkephalin and, hence, cannot confirm these findings.

We showed that PV distributes moderately to the ento-rhinal cortex and to the ventral subiculum of thehippocampus—mainly to the rostral EC/subiculum and toventral aspects of the subiculum, adjoining EC. Previousreports (Berendse and Groenewegen, 1991; Moga et al.,1995) have similarly described PV projections to EC and tothe ventral subiculum and, like here, stronger projectionsfrom the anterior than posterior PV. Retrograde tracerinjections in the hippocampus, involving the ventral sub-iculum, give rise to labeled cells in PV (mainly PVa) (Wysset al., 1979; Riley and Moore, 1981; Su and Bentivoglio,1990), and the hippocampus is the source of significantreturn projections to PV, originating from the ventralsubiculum (Witter, 2006).

As described, the amygdala is a major PV target, withprojections heaviest to the central (CEA) and basomedial(BMA) nuclei of amygdala. While PV projections to BLAare less dense than to CEA and BMA, they are nonethe-less pronounced, mainly targeting the posterior BLA(BLAp), bordering BMA. Earlier reports have similarlydescribed marked PV projections to BLA (Ottersen andBen-Ari, 1979; Berendse and Groenewegen, 1990, 1991;Su and Bentivolglio, 1990; Turner and Herkenham, 1991;Moga et al., 1995). BLA is an integral part of the “prelim-bic circuit.” BLA has strong links with PL (Sesack et al.,1989; McDonald, 1987, 1991; McDonald et al., 1996;Conde et al., 1995; Vertes, 2004; Gabbott et al., 2006;Hoover and Vertes, 2007), as well as with other parts ofthe circuit including the hippocampus, ACC, claustrum,and the insular cortex (McDonald, 1987; Brog et al., 1993;Petrovich et al., 1996; Pikkarainen et al., 1999; Majak etal., 2002).

PV as an interface in the flow of information between

the suprachiasmatic nucleus (SCN) and other regions

of the brain. In accord with previous reports (Moga etal., 1995; Moga and Moore, 1997; Krout et al., 2002), weshowed that PV projects moderately densely to the supra-chiasmatic nucleus of the hypothalamus. SCN, in turn, isthe source of significant projections to PV (Watts et al.,1987; Novak et al., 2000; Peng and Bentivoglio, 2004;Zhang et al., 2006). Accordingly, PV appears to representan important relay in the transfer of information to andfrom the SCN—the circadian pacemaker (Mistlberger,2005; Morin and Allen, 2006). While afferents to SCNgenerally serve to entrain SCN activity to light/dark con-ditions, PV lesions do not disrupt circadian timing orentrainment to light (Ebling et al., 1992). This suggests anonphotic modulatory influence of PV on SCN. Moga et al.(1995) proposed that PV conveys information on basallevels of activation to the SCN—functions associated withPV/midline thalamus (Van der Werf et al., 2002; Vertes,2006).

Regarding SCN-PV projections, the SCN has few directoutputs to the systems it affects (Deurveilher and Semba,2005; Morin and Allen, 2006), indicating indirect routes tothem, possibly through PV. At the light and EM levels,Peng and Bentivoglio (2004) showed that SCN stronglytargets PV, and further that SCN fibers synapse with PVcells projecting to the amygdala. On this basis they con-cluded that PV “plays a role in the transfer of circadiantiming information to the limbic system.”



Figure 13



PT projections: comparisons with previousstudies

As discussed, there is significant overlap in PV and PTprojections. Although a few reports have described PTprojections to specific targets, to our knowledge no previ-ous study has examined the totality of PT projections.

We showed that PT distributes densely throughout theventral PFC to AC, PL, IL, and medial orbital (MO) cor-tices, with projections heaviest to PL. In an early exami-nation of midline and intralaminar thalamic connectionswith the cortex, Berendse and Groenewegen (1991) simi-larly described PT projections to the mPFC, but unlikehere, projections were modest and largely confined to ven-tral aspects of the mPFC, mainly to MO and IL. Their PTinjection, however, was small and restricted to the medialaspect of the anterior PT (see their fig. 1A, p. 75; Berendseand Groenewegen, 1991). Supporting present findings,retrograde tracer injections in AC, PL, and IL have beenshown to give rise to significant numbers of labeled cells inPT (Conde et al., 1995; Hoover and Vertes, 2007). PT alsostrongly targets the ventral mPFC in primates, mainly IL(area 25) and PL (area 32) (Hsu and Price, 2007).

Similar to PV, ACC is a major destination of PT fibers.In general accord with present results, two early studies(Kelley and Stinus, 1984; Carlsen and Heimer, 1986) de-scribed robust PT projections to ACC, terminating heavilyin the medial two-thirds of ACC (shell region) with someextension dorsally into dorsomedial aspects of CP. Be-rendse and Groenewegen (1990) confirmed marked PTprojections to the shell of ACC, particularly dense to theventromedial shell of ACC. Although they also reportedthat PT distributes to the core of ACC and to the dorso-medial CP, projections were considerably less pronouncedthan the presently described massive distribution of PTfibers to the core of ACC and to the dorsomedial CP (seeFigs. 7D–F, 10A). Consistent with our results, Brog et al.(1993) described significant numbers of labeled cells in PTfollowing retrograde tracer injections in the core or shell ofACC.

As demonstrated, PT fibers mainly target caudal re-gions of the amygdala, predominantly the lateral andbasal nuclei of amygdala. In contrast to the dense PVinnervation of CEA, PT fibers largely avoid the core of

CEA, projecting instead to the fringes of CEA. Ottersenand Ben-Ari (1979) identified few labeled cells in PT withretrograde injections in CEA, the cortical nuclei, or ante-rior regions of the basal nuclei, but described significantnumbers of reacted cells in PT following large amygdalarinjections spanning the rostral and caudal BMA/BLA.Subsequent reports using retrograde (Su and Bentivoglio,1990) or anterograde tracers (Turner and Herkenham,1991) similarly demonstrated PT projections to the lat-eral, basomedial, and basolateral nuclei of amygdala.

We showed that PT distributes throughout entorhinalcortex and the ventral subiculum, terminating withinfairly restricted zones of both sites: mainly inner layers(3–6) of the lateral EC and the molecular layer of theventral subiculum. Berendse and Groenewegen (1991)demonstrated a similar distribution of PT fibers to EC andto the ventral subiculum, but in contrast to the presentfindings described stronger PV than PT projections tothese sites—we found the opposite. Differences could in-volve relative size and placements of injections in PT andPV. Several reports have identified labeled cells in PTfollowing retrograde tracer injections in the hippocampus(Wyss et al., 1979; Riley and Moore, 1981; Su and Ben-tivoglio, 1990), or entorhinal cortex (Beckstead, 1978;Wyss et al., 1979; Insausti et al., 1987).

Functional considerations

Although the projections of PV and PT significantlyoverlap, suggesting comparable functions, considerablygreater attention has been given to the functional charac-teristics of PV. An accumulating body of evidence indi-cates that PV receives inputs from several sites of thebrainstem and hypothalamus that are known to exertactivating and/or ‘wakefulness-promoting’ effects on theforebrain. This includes afferents from monoaminergic,cholinergic, and peptide-containing systems of the brain-stem and diencephalon, prominently including orexin/hypocretin cells of the lateral hypothalamus (Chen andSu, 1990; Vertes, 1991; Freedman and Cassell, 1994;Otake and Ruggiero, 1995; Peyron et al., 1998; Cutler etal., 1999; Vertes et al., 1999; Bhatnagar et al., 2000; Kroutet al., 2002; Kirouac et al., 2005; Otake, 2005; Parsons etal., 2006). Accordingly, PV/PT (and other nuclei of themidline thalamus) are thought to serve an essential role inarousal and attention (Van der Werf et al., 2002; Vertes,2006; Vertes et al., 2006).

In line with the foregoing, PV cells show elevated levelsof c-fos expression during wakefulness (Peng et al., 1995;Novak et al., 2000) as well as during stressful conditions,elicited by various stressors (Chastrette et al., 1991; Bub-ser and Deutch, 1999; Sica et al., 2000; Otake et al., 2002).PV appears to be critically involved in adaptive responsesto stress (Bhatnagar and Dallman, 1998; Bhatnagar et al.,2000, 2002; Otake et al., 2002) through direct (Sawchenkoand Swanson, 1983; present results), or predominantlyindirect projections to paraventricular nucleus of the hy-pothalamus (Sawchenko and Swanson, 1983; Bhatnagarand Dallman, 1998; Dong et al., 2001; Otake et al., 2002;Dong and Swanson, 2006).

In addition to its role in promoting arousal/wakefulness,the orexin system participates in feeding behavior (forreview, see Willie et al., 2001). Intraventricular injectionsof orexin (orexin A) stimulates food consumption in sati-ated rats (Sakurai et al., 1998; Edwards et al., 1999;Haynes et al., 1999), anti-orexin antibodies or receptor

Fig. 13. A–C: Series of low-magnification brightfield photomicro-graphs of transverse sections through the forebrain depicting the siteof a FluoroGold injection in the basolateral nucleus (BLA) of theamygdala (A) and patterns of retrogradely labeled cells within theanterior paraventricular (PV) and paratenial (PT) nuclei (B) and theposterior paraventricular nucleus (C) produced by this injection. Notesignificant numbers of retrogradely labeled neurons in the posteriorPV but relatively few numbers in the anterior PV and PT with thisinjection. D–F: Series of low-magnification brightfield photomicro-graphs of transverse sections through the forebrain depicting the siteof a FluoroGold injection in the central nucleus (CEA) of the amygdala(D) and patterns of retrogradely labeled cells within the anteriorparaventricular (PV) and paratenial (PT) nuclei (E) and the posteriorparaventricular nucleus (F) produced by this injection. Note moderatenumbers of retrogradely labeled cells in anterior and posterior PV,and relatively few in PT. Finally, note the presence of labeled neuronsin other nuclei of the midline thalamus (C,F) produced with BLA andCEA injections; namely, in the intermediodorsal and central medialnuclei (C) and the rhomboid and reuniens nuclei (F). Scale bar � 750�m for A; 300 �m for B; 500 �m for C; 700 �m for D; 400 �m for E; 450�m for F. See list for abbreviations.



antagonists suppress feeding (Haynes et al., 2000), andorexin knockout mice are hypophagic (Hara et al., 2001).These effects may in part be mediated the actions of orexinon PV. Specifically, Nakahara et al. (2004) described ele-vated c-fos expression in PV in anticipation of feeding(food anticipatory activity) in deprived rats, and furtherreported that PV lesions attenuated anticipatory locomo-tor activity associated with feeding. Angeles-Castellanoset al. (2007) similarly reported elevated c-fos expression inPV with anticipated feeding and, interestingly, also de-scribed enhanced c-fos expression in several other limbicforebrain structures before and immediately after the de-livery of food to deprived rats, including the central andbasal nuclei of the amygdala, BST, the lateral septum,ACC (core and shell) and the infralimbic/prelimbic corti-ces. As demonstrated here, these are the main forebraintargets of PV (and PT), suggesting a PV/PT influence onthem in complex behaviors associated with feeding.

Related to this, Kelley et al. (2005) recently put forth amodel suggestive of a role for PV in motivated behaviorsas exemplified by feeding. Although not necessarily lim-ited to feeding, the model addressed mechanisms respon-sible for the ingestion of palatable foods under satiatedconditions. According to Kelley et al. (2005), PV receivesdiverse inputs which, among other things, code the incen-tive value of foods and when incentives are high (desirablefoods), feeding ensues, even in a satiated state, mainlythrough the actions of PV on the ventral striatum. Theystated: “PVT may act as an interface between signalsrelated to arousal, energy balance, circadian or diurnalrhythms, and reward, and major striatal motor outputsystems.”

In summary, PV (and PT) receive a vast array of affer-ents from the brainstem, hypothalamus, and limbic fore-brain and appear to serve as a critical gateway for thetransfer of multimodal information to structures of thelimbic system in the selection of appropriate responses tochanging environmental conditions. In effect, dependingon the relative dominance of sets of inputs to PV/PT,coupled with their output to specific structures of thelimbic forebrain, PV/PT would guide behavior toward aparticular outcome among various potential outcomes.

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