Efferent Projections of Reuniens and Rhomboid Nuclei of the Thalamus
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THE JOURNAL OF COMPARATIVE NEUROLOGY 499:768 –796 (2006) 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. RODRIGUEZ1,3 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 Phaseolus 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 longitudinally 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 widespread regions of the cortex (recruiting responses; Dempsey © 2006 WILEY-LISS, INC. 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: vertes@ccs.fau.edu 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. DOI 10.1002/cne EFFERENTS OF REUNIENS AND RHOMBOID NUCLEI 769 Groenewegen and Berendse, 1994). The notion of the midline thalamus as “nonspecific” has been revised, however, based in large part on recent anatomical findings showing that as a group the midline nuclei project not widely throughout the neocortex but, rather, selectively to specific regions of the prefrontal cortex (Berendse and Groenewegen, 1991; Van der Werf et al., 2002; Vertes et al., 2003; Groenewegen and Witter, 2004). It is well established that nucleus reuniens (RE) distributes densely to the hippocampal formation (HF; Herkenham, 1978; Wouterlood et al., 1990; Wouterlood, 1991; Bokor et al., 2002). RE axons form asymmetric (excitatory) contacts predominantly on distal dendrites of pyramidal cells in stratum lacunosum-moleculare of CA1 and the subiculum (Wouterlood et al., 1990). RE stimulation produces 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 prefrontal 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 mPFC to the hippocampus (Goldman-Rakic et al., 1984; Room et al., 1985; Reep et al., 1987; Sesack et al., 1989; Hurley et al., 1991; Takagishi and Chiba, 1991; Buchanan et al., 1994). The recent demonstration that mPFC strongly targets RE (Vertes, 2002, 2004), coupled with direct RE to HF projections (Herkenham, 1978; Wouterlood et al., 1990; Wouterlood, 1991; Bokor et al., 2002), suggests that RE is the main route for the actions of the mPFC on the hippocampus/parahippocampus. This system of connections (mPFC-RE-HF) thus completes an important functional loop between HF and mPFC. RE receives widespread, mainly limbic, input from the brainstem, hypothalamus, amygdala, basal forebrain, and limbic cortex (Herkenham, 1978; Risold et al., 1997; McKenna and Vertes, 2004). RE is pivotally positioned to relay a vast array of (limbic) information to its main targets. Abbreviations AC ACC ACo AD AGl AGm AH AI,d,p,v AM AON APN AV BLA BLAa,p BMA BST CA1,2,3 CB CC CEA CEM CLA COA CP DBh DG DMh EC,l,m ECld,v ECT EN FP,l,m GI GP HF IAM IL IMD LA LD LG,d LH LHy LO LP LS LV MA MB MD anterior cingulate cortex nucleus accumbens anterior commissure anterodorsal nucleus of thalamus lateral agranular (frontal) cortex medial agranular (frontal) cortex anterior nucleus of hypothalamus agranular insular cortex, dorsal, posterior, ventral divisions anteromedial nucleus of thalamus anterior olfactory nucleus anterior pretectal nucleus anteroventral nucleus of thalamus basolateral nucleus of amygdala BLA anterior, posterior divisions basomedial nucleus of amygdala bed nucleus of stria terminalis field CA1, CA2, CA3 of Ammon’s horn cinguum bundle corpus callosum central nucleus of amygdala central medial nucleus of thalamus claustrum cortical nucleus of amygdala caudate-putamen nucleus of the diagonal band, horizontal limb dentate gyrus of hippocampus dorsomedial nucleus of hypothalamus entorhinal cortex, lateral, medial divisions ECl, dorsal, ventral parts ectorhinal cortex endopiriform nucleus frontal polar cortex, lateral, medial divisions granular insular cortex globus pallidus hippocampal formation interanteromedial nucleus of thalamus infralimbic cortex intermediodorsal nucleus of thalamus lateral nucleus of amygdala lateral dorsal nucleus of thalamus lateral geniculate nucleus, dorsal division lateral habenula lateral hypothalamic area lateral orbital cortex lateral posterior nucleus of thalamus lateral septal nucleus lateral ventricle magnocellular preoptic nucleus mammillary bodies mediodorsal nucleus of thalamus MFB MO mPFC MPO MRF MS MT OC OT PA PAG PARA PC PH PIR PL PO POST PPC PRC pRE PRE PT PV PVh RE RF RH RSC RSCagl,d RT SF SI slm sm SMT SSI SSII SUB,d,v SUM TE TT,d,v VAL VB VLO VM VMh VO VTA ZI 3V medial forebrain bundle medial orbital cortex medial prefrontal cortex medial preoptic area mesencephalic reticular formation medial septum mammillothalamic tract occipital cortex olfactory tubercle piriform-amygdaloid transition area periaqueductal gray parasubiculum of HF paracentral nucleus of thalamus posterior nucleus of hypothalamus piriform cortex prelimbic cortex posterior nucleus of thalamus postsubiculum of HF hippocampus posterior parietal cortex perirhinal cortex perireuniens nucleus presubiculum of HF paratenial nucleus of thalamus paraventricular nucleus of thalamus paraventricular nucleus of hypothalamus nucleus reuniens of thalamus rhinal fissue rhomboid nucleus of thalamus retrosplenial cortex lateral agranular, dorsal fields of RSC reticular nucleus of thalamus septofimbrial nucleus substantia innominata stratum lacunosum-moleculare of Ammon’s horn stria medullaris submedial nucleus of thalamus primary somatosensory cortex secondary somatosensory cortex subiculum, dorsal, ventral parts supramammillary nucleus temporal cortex tenia tecta, dorsal, ventral parts ventral anterior-lateral complex of thalamus ventrobasal complex of thalamus ventrolateral orbital cortex ventral medial nucleus of thalamus ventromedial nucleus of hypothalamus ventral orbital cortex ventral tegmental area zona incerta third ventricle The Journal of Comparative Neurology. DOI 10.1002/cne 770 R.P. VERTES ET AL. Although RE projections to HF have been well documented (Herkenham, 1978; Wouterlood et al., 1990; Su and Bentivoglio, 1990; Wouterlood, 1991; Dolleman-Van der Weel and Witter, 1996; Bokor et al., 2002), few reports have 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. Specifically, as with RE, RH receives afferents from widespread regions of the brainstem and forebrain (Van der Werf et al., 2002; Vertes et al., 2004b; Owens, 2005), projects to the hippocampus (Riley and Moore, 1981; Su and Bentivoglio, 1990), and exerts excitatory actions on HF (Dolleman-Van der Weel et al., 1997; Bertram and Zhang, 1999). Very little is known about the connections of RH, either inputs or outputs. To our knowledge, only a single early report concerning the rat (Ohtake and Yamada, 1989) has examined RH projections. Based on the pivotal positions of RE and RH in the limbic circuitry and the general lack of information on their connections, we sought to examine comprehensively the efferent projections of the reuniens and rhomboid nuclei of the midline thalamus. We found that RE distributes densely to the orbitomedial prefrontal cortex, hippocampus/parahippocampus, and some subcortical limbic sites. RE projections are more restricted than those of RH. RE projects much more strongly than RH to the hippocampus and to the entorhinal cortex, whereas RH distributes more heavily than RE to nucleus accumbens. Based on their efferent projections, RE and RH are strategically positioned to influence, and possibly coordinate, the activity of select limbic subcortical and cortical structures. treated with 1% sodium borohydride in 0.1 M phosphate buffer (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 in 0.5% bovine serum albumin in 0.1 M Tris-buffered saline (TBS; pH, 7.6) at room temperature (RT) to minimize nonspecific labeling. After this, sections were incubated overnight at RT in 0.1% bovine serum albumin in TBS containing 0.25% Triton X-100 and biotinylated goat antiPHA-L (Vector, Burlingame, CA) at a dilution 1:500. Sections were then washed in PBS (5 ⫻ 5 minutes) and placed 1) for 1 hour in 1:400 dilution of biotinylated rabbit antigoat immunoglobulin (IgG) and 2) for 1 hour in 1:200 dilution of peroxidase-avidin complex from the Vector Elite kit. After 5 ⫻ 5 minute rinses, sections were incubated in a solution containing 0.022% of 3,3⬘ diaminobenzidine (DAB) in PBS for 5 minutes, followed by a second 5-minute DAB (same concentration) incubation to which 0.003% H2O2 had been added. Sections were then rinsed again in PBS (3 ⫻ 1 minutes) and mounted onto chromealum-gelatin-coated slides. An adjacent series of sections from each rat was stained with cresyl violet for anatomical reference. Sections were examined via light- and darkfield optics. Injection sites and labeled fibers were plotted on representative schematic transverse sections through the brain with sections adapted from the rat brain atlas of Swanson (1998). Material judged particularly useful for emphasizing or clarifying points of text was illustrated with light- and darkfield photomicrographs with a Nikon DXM1200 digital camera mounted on a Nikon Eclipse microscope. Digital images were captured and reconstructed in ImagePro and enhanced (contrast and brightness) in Adobe Photoshop 9.0. 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 275–325 g. These experiments were approved by the Florida Atlantic University Institutional Animal Care and Use Committee and conform to all Federal regulations and National Institutes of Health guidelines for the care and use of laboratory animals. Powdered lectin from PHA-L was reconstituted to 5% in 0.05 M sodium phosphate buffer, pH 7.4. The PHA-L solution was iontophoretically deposited in the brains of anesthetized rats by means of a glass micropipette with an outside tip diameter of 40 – 60 m. Positive direct current (5–10 A) was applied through a Grass stimulator (model 88) coupled with a high-voltage stimulator (FHC, Bowdoinham, ME) at 2 seconds “on”/2 seconds “off” intervals for 30 – 40 minutes. After a survival time of 7–10 days, animals were deeply anesthetized with sodium pentobarbital and perfused transcardially with a buffered saline wash (pH 7.4, 300 ml/animal), followed by fixative (2.5% paraformaldehyde, 0.2– 0.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4; 300 –500 ml/animal) and then by 10% sucrose in the same phosphate buffer (150 ml/ animal). The brains were removed and stored overnight at 4°C in 30% sucrose in the same phosphate buffer. Brains were generally cut on the following day, and 40- or 50-m frozen sections were collected in phosphate-buffered saline (PBS; 0.9% sodium chloride in 0.1 M sodium phosphate buffer, pH 7.4). A complete series of sections was RESULTS The patterns of distribution of labeled fibers throughout the brain with injections in the reuniens and rhomboid nuclei of the midline thalamus are described. Figure 1 depicts sites of injections in RE (Fig. 1A,B) and RH (Fig. 1C,D) for the schematically illustrated cases. The patterns of labeling obtained with the schematically depicted cases are representative of patterns seen with nonillustrated cases. RE and RH cases are compared with control cases with injections in the central medial nucleus (CEM) of the thalamus, dorsal to RH. RE (proper) cases are also compared with cases with injections in the perireuniens region (or the wings of RE; Risold et al., 1997, Swanson, 1998). Figure 2 shows cytoarchitectural boundaries of nuclei of the midline thalamus at four levels of the thalamus (Fig. 2A–D) together with schematic depictions of representative 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 distribution of labeled fibers following a PHA-L injection in RE (case RE-20; Figs. 1A, 2F–H). Labeled fibers coursed primarily ventrolaterally from RE, traversing the ventromedial nucleus of thalamus, the zona incerta, and the dorsolateral hypothalamus to reach the medial forebrain bundle (MFB; Fig. 3P–S) and from there followed rostral, caudal, or lateral routes. The bulk of labeled axons as- The Journal of Comparative Neurology. DOI 10.1002/cne EFFERENTS OF REUNIENS AND RHOMBOID NUCLEI 771 Fig. 1. A,C: Low-power lightfield photomicrograph showing the sites of Phaseolus vulgaris leuccoagglutinin (PHA-L) injections in the reuniens (A; case RE-20) and rhomboid nuclei (C; case RH-7) of the midline thalamus. B,D: High-magnification lightfield photomicro- graphs from the core of injections depicting patterns of PHA-L-filled cells in RE (B) and RH (D). For abbreviations see list. Scale bar ⫽ 1,000 m for A,C; 175 m for B,D. cended through the lateral hypothalamus/MFB to the basal forebrain, where they joined the internal capsule coursing through ventromedial regions of the striatum in discrete fascicles to the rostral forebrain (Fig. 3G–L). At the anterior forebrain, they either distributed terminally to parts of the frontal cortex or turned caudally, coursing through the cingulum bundle to the hippocampus or through outer layers of the frontal and then caudal re- gions of cortex. A second prominent bundle exited laterally from the lateral hypothalamus bound for the amygdala and ventrolateral regions of cortex bordering the rhinal fissure. Some labeled fibers of this tract continued caudally to reach parts of the subiculum of hippocampus. The smallest of the three bundles descended through lateral hypothalamus/MFB to caudal regions of the diencephalon (mainly to the hypothalamus) and to the rostral midbrain. Fig. 2. A–D: Series of Nissl-stained sections through the thalamus showing cytoarchitectural boundaries of nuclei of the midline thalamus and surrounding structures. E–H: Series of matched (to stained sections, A–D) schematic sections through the thalamus showing the locations 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 central medial nucleus of thalamus (case CEM-10). For abbreviations see list. Scale bar ⫽ 1,000 m for A–D. The Journal of Comparative Neurology. DOI 10.1002/cne EFFERENTS OF REUNIENS AND RHOMBOID NUCLEI 773 TABLE 1. Density of Labeling in Forebrain Produced by PHA-L Injections in the Reuniens and Rhomboid Nuclei of the Midline Thalamus1 Labeling Structures Telencephalon Cortex Cingulate Ectorhinal Entorhinal Medial Lateral Frontal polar Medial Lateral Infralimbic Insular Dorsal agranular Ventral agranular Posterior agranular Dysgranular Granular Lateral agranular (motor) Medial agranular (motor) Occipital Orbital Lateral Medial Ventral Ventrolateral Perirhinal Piriform Anterior part Posterior part Prelimbic Retrosplenial Somatosensory I Somatosensory II Temporal Accumbens n. Shell Core Amygdala Anterior area Basolateral Basomedial Central Capsular part Medial part Cortical Anterior part Posterior part Medial Lateral Posterior Anterior olfactory n. Medial part Ventral part Bed n. of stria terminalis Caudate-putamen Claustrum Diagonal band n. Horizontal limb Vertical limb Endopiriform n. Globus pallidus Hippocampal formation CA1 1 Labeling RE RH ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ — ⫹⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹ ⫹ ⫹⫹ ⫹⫹ — ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹ ⫹ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹ ⫹ ⫹ ⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹ — ⫹ ⫹ ⫹ ⫹⫹⫹ ⫹⫹ — — ⫹ ⫹ — ⫹ ⫹ ⫹ ⫹ — ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹⫹⫹ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹ ⫹ — — ⫹ ⫹ ⫹⫹ — ⫹⫹⫹ ⫹⫹⫹ Structures Dentate gyrus Subiculum Lateral septum Dorsal n. Intermediate n. Ventral n. Lateral preoptic area Magnocellular preoptic n. Medial preoptic area Median preoptic n. Medial septal n. Olfactory tubercle Septofimbrial n. Septohippocampal n. Substantia innominata Tania tecta Dorsal Ventral Diencephalon Thalamus Anterodorsal n. Anteromedial n. Anteroventral n. Central lateral n. Central medial n. Interanteromedial Intermediodorsal n. Lateral geniculate n. Lateral habenula Llaterodorsal n. Lateroposterior n. Medial geniculate n. Medial habenula Mediodorsal n. Medial division Central division Lateral division Paracentral n. Parafascicular n. Paratential n. Paraventricular n. Anterior part Posterior part Posterior n. Reticular n. Reuniens n. Rhomboid n. Submedial n. Ventral anterior-lateral n. Ventral basal copmplex Hypothalamus Anterior n. Dorsal hypothalamic area Dorsomedial n. Lateral n. Mammillary bodies Paraventricular n. Posterior n. Supramammillary n. Subthalamus Zona incerta RE RH — ⫹⫹⫹ — ⫹⫹⫹ — ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ — — ⫹ — ⫹⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹⫹ — — ⫹⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹ — ⫹ ⫹ — ⫹ ⫹ ⫹ — ⫹ — — — — — ⫹ ⫹ — ⫹ ⫹ ⫹ — ⫹ — — — — ⫹ ⫹ — — — ⫹ ⫹ ⫹ — — — — ⫹ ⫹ — ⫹ ⫹ ⫹ ⫹ — — ⫹ ⫹ — ⫹ ⫹ ⫹ ⫹ — — — — — ⫹ ⫹ — — ⫹ — — — ⫹ — — ⫹ ⫹ ⫹ ⫹ ⫹, Light labeling; ⫹⫹, moderate labeling, ⫹⫹⫹, dense labeling; —, absence of labeling; n, nucleus; PHA-L, Phaseolus vulgaris-leucoagglutinin; for other abbreviations see list. Labeling within anterior levels of the forebrain (preseptum; Fig. 3A–H). As illustrated in Figure 3A–H, labeling was prominent within the rostral forebrain. At the anterior pole of the forebrain (Figs. 3A–D, 4), labeling was heavy within: 1) the medial frontal polar (FPm), prelimbic (PL), medial orbital (MO), ventral orbital (VO), ventrolateral orbital (VLO), and anterior piriform (PIR) cortices (Fig. 4B,D); 2) the dorsal tenia tecta (TTd; Fig 4B,D); and 3) the claustrum (CLA). Labeling was present but less dense in the anterior cingulate (AC) and medial (frontal) agranular (AGm) cortices and was prominent within layer 1 of these cortical fields (Figs. 3C,D, 4A,C). More caudally in the rostral forebrain (Fig. 3E–H), labeling remained strong dorsoventrally within the mPFC, stronger ventrally in the infralimbic (IL) and prelimbic cortices than dorsally in AGm and AC, and heavily concentrated in layers 1 and 5/6 of IL and PL (Figs. 3E–H, 5A–C). Labeling was also pronounced within the ventral agranular insular cortex (AIv), IL, TTd, and CLA and light to moderate within the lateral (frontal) agranular cortex (AGl), PIR, olfactory tubercle (OT), and medial parts of the striatum (Fig. 3E–H). A dense cluster of (terminally) labeled fibers was present in the rostral pole of nucleus accumbens (ACC; Figs. 3F, 5A,C), but few fibers were seen The Journal of Comparative Neurology. DOI 10.1002/cne 774 R.P. VERTES ET AL. Fig. 3. Schematic representation of labeling present in select sections through the forebrain and diencephalon (A–U) and ventral hippocampus (AA–II) produced by a PHA-L injection (dots in Q–S) in nucleus reuniens (case RE-20). Sections modified from the rat atlas of Swanson (1998). For abbreviations see list. in other regions of ACC (Figs. 3G–K, 5B). For the most part, the labeling of the dorsal striatum (CP) involved fibers of passage to the anterior forebrain. There was a virtual absence of labeling in lateral regions of the prefrontal/frontal cortex within the primary motor (AGl) and sensory/somatosensory cortices. The Journal of Comparative Neurology. DOI 10.1002/cne EFFERENTS OF REUNIENS AND RHOMBOID NUCLEI Figure 3 775 (Continued) The Journal of Comparative Neurology. DOI 10.1002/cne 776 R.P. VERTES ET AL. Figure 3 Labeling within midlevels of the forebrain (anterior septum to rostral hippocampus; Fig. 3I–P). At midlevels of the anterior forebrain (Fig. 3I–P), labeling was largely confined to cortical sites, TTd, and claustrum. Within the cortex (Fig. 3I–L), relatively significant numbers of labeled fibers were present: 1) dorsomedially in AC and AGm and, as rostrally, heavily concentrated in layers 1, 5, and 6 of these fields and 2) ventrolaterally in the insular cortex, extending from the dorsal and ventral agranular insular fields (AId and AIv) caudally to the posterior agranular insular cortex (AIp). Labeled fibers spread quite uniformly throughout all layers of the insular cortex. In addition, AGl (primarily outer layers) was moderately labeled, and parts of the basal forebrain, including (Continued) the medial and lateral septum and diagonal band nuclei, were lightly labeled. More caudally (Fig. 3M–P), labeled fibers were localized mainly to specific dorsomedial and ventrolateral parts of the cortex and to CLA, with few present in other regions of cortex or subcortically. Specifically, labeling was pronounced 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 the somatosensory cortex, largely confined to layer 1 of the lateral convexity of cortex, appeared mainly to traverse these regions in route to parahippocampal cortices. Subcortically, the magnocellular preoptic nucleus, lateral pre- The Journal of Comparative Neurology. DOI 10.1002/cne EFFERENTS OF REUNIENS AND RHOMBOID NUCLEI Fig. 4. A,B: Low-magnification darkfield photomicrographs of transverse sections through the rostral forebrain showing patterns of labeling within the orbitomedial prefrontal cortex produced by an injection in nucleus reuniens (case 20). C,D: High-magnification darkfield photomicrographs taken from B (arrowheads). Note the presence 777 of pronounced numbers of labeled fibers along the medial wall of the medial prefrontal cortex (densely concentrated in layers 1 and 5/6) as well 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. For abbreviations see list. Scale bar ⫽ 1,000 m for A,B; 250 m for C,D. Fig. 5. A,B: Low-magnification darkfield photomicrographs of transverse sections through the rostral forebrain showing patterns of labeling within the orbitomedial prefrontal cortex produced by an injection in nucleus reuniens (case RE-20). Note the presence of pronounced 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 ventral agranular insular cortex. C: High-magnification darkfield photomicrograph from A (arrowheads) showing labeled fibers in all layers of IL, PL, and AC, densely concentrated in layers 1 and 5/6, and a mediolateral orientation within middle layers of these fields parallel to the cell layers. For abbreviations see list. Scale bar ⫽ 1,000 m for A,B; 250 m for C. The Journal of Comparative Neurology. DOI 10.1002/cne EFFERENTS OF REUNIENS AND RHOMBOID NUCLEI 779 optic area, paraventricular nucleus of thalamus, and lateral 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), labeling within the (iso-/allo-) cortex was confined mainly to dorsomedial and ventrolateral parts of cortex; that is, to the dorsal and lateral agranular RSC (RSCd and RSCagl; Risold et al., 1997) dorsomedially, and to the perirhinal cortex (PRC; particularly inner layers) and to lesser degree to the ectorhinal (ECT) and the lateral entorhinal (EC) cortices, bordering PRC, ventrolaterally. Labeled fibers on the lateral convexity of cortex (Fig. 3Q–U) appeared largely bound for ECT, PRC, and EC. At these same levels (Fig. 3R–U), a dense band of labeled fibers was present throughout stratum lacunosum-moleculare (slm) of CA1 of the dorsal hippocampus (Figs. 3R–U, 6A). There was an absence of labeling in CA2 or CA3 of Ammon’s horn or in the dentate gyrus (Fig. 6A). Subcortically, the mediodorsal and central medial nuclei of thalamus, the supramammillary nucleus of the hypothalamus and the medial, anterior cortical, lateral, and basolateral nuclei of amygdala were lightly labeled. Labeling with the ventral hippocampus (Fig. 3AA– HH). As discussed, labeled fibers reached the ventral hippocampus, subiculum, and parahippocampal regions mainly through the cingulum bundle (CB; Figs. 3AA,BB, 6A,B) and secondarily through lateral cortical routes, that is, through the dorsomedial PFC, around the lateral convexity of cortex (mainly within layer 1) to ventrolateral regions of cortex and parts of the subiculum. As depicted in Figure 3AA–HH, the ventral hippocampus and associated parahippocampal regions were densely labeled. Labeled fibers were abundantly present throughout the slm of CA1, the dorsal and ventral subiculum, the pre- and parasubiculum, 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 ventral hippocampus was heavily labeled (Fig. 3AA–HH). This is depicted at two levels of the ventral hippocampus in the photomicrographs in Figure 6B,C. Labeling was equally pronounced throughout the molecular layer of the ventral subiculum, continuous caudally with slm of CA1 (Figs. 3DD–FF, 6C), and significant but somewhat less dense in the pre- and parasubiculum (heaviest in layer 1) and inner layers of the postsubiculum (Figs. 3DD–HH, 7A). Within the parahippocampus, dense collections of labeled fibers were observed rostrocaudally throughout ECT and PRC, mainly confined to the region around the rhinal fissure, rostrally (Fig. 3R–FF), with some extension to dorsal regions of ECT, caudally (Fig. 3GG,HH). Although strongest in layer 1, labeling was present throughout all layers of ECT and PRC (Figs. 6C, 7A). Within the EC, labeling was considerably stronger 1) in the ECl than the medial EC (ECm), 2) in the dorsal than the ventral divisions of ECl, and 3) in the caudal than the rostral parts of the dorsal ECl. Specifically, beginning approximately at the level of the fusion of the dorsal and ventral CA1 of the hippocampus (Fig. 3BB) and continuing throughout the caudal extent of EC, the entire expanse (all layers) of the dorsal 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, but little 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) than in the dorsal ECl. This is depicted in Figure 7B–D, showing dense aggregates of labeled fibers in ECm and EClv at three levels of ventral hippocampus produced by a rostral RE injection (case aRE 9, Fig. 2E,F). This contrasts with an essential absence of ECm and EClv labeling (Fig. 7A) with a caudal RE injection (Figs. 1A, 2F–H). Nucleus perireuniens Extending laterally from the main body of RE, for approximately the caudal half of RE, are the “lateral wings” or the perireuniens region, or nucleus perireuniens (pRE; Risold et al., 1997; Van der Werf et al., 2002). Injections within pRE gave rise to a pattern of labeling similar to that observed with injections in RE (proper), with some significant differences. As expected, a notable difference was that labeling with pRE injections was predominantly unilateral (ipsilateral), as opposed to evenly distributed on both sides of the brain with RE (proper) injections. At best, a few labeled fibers were observed contralaterally with pRE injections in ventrolateral regions of the cortex and in CA1 and subiculum of HF. Similar to RE, labeling was more pronounced cortically than subcortically and was restricted primarily to “limbic” regions of cortex, including the orbitomedial, insular (anterior and posterior divisions), ECT, PRC, EC (mainly ECl), and CA1/subiculum of HF. For the most part, fewer labeled fibers were observed in commonly labeled sites with pRE than with RE injections. This was particularly the case subcortically within ACC and CLA. A few regions, however, showed stronger labeling with pRE compared with RE injections. The most notable of these were the ventral and ventrolateral orbital cortices (VO, VLO), ECl, and ventral subiculum. Specifically, layers 1 and 3 of VO and VLO and to lesser extent LO (Fig. 8A), the longitudinal extent of ECl immediately ventral to the rhinal fissure (Fig. 8B,C), and the ventral subiculum (Fig. 8B,C) were densely labeled with pRE injections. Rhomboid nucleus: case RH-7 The site of injection for the schematically illustrated RH case (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 distribution of labeled fibers after this injection. Similar to RE cases, the bulk of labeled fibers coursed ventrolaterally from RH (Fig. 9P–R) and at the exit level from RH, split into two main bundles: one ascending to the rostral forebrain in the general region of the MFB, the other coursing laterally to parts of the amygdala and parahippocampal cortices. At the caudal septum, some labeled axons of the ascending bundle continued forward to parts of the basal forebrain (Fig. 9F–N); the majority, however, turned dorsolaterally into the striatum to join the internal capsule, coursing medially/dorsomedially through the striatum to the anterior forebrain. At the rostral forebrain, fibers of this bundle spread terminally to regions of the frontal cortex or swung caudally coursing within the cingulum bundle or through lateral parts of the cortex to posterior regions of cortex and to the hippocampus. The secondary bundle exited laterally from RH, primarily bound for the amygdala, parahippocampal cortices, and ventral subiculum. The Journal of Comparative Neurology. DOI 10.1002/cne Fig. 6. Low-magnification darkfield photomicrographs showing patterns of labeling in the dorsal (A) and ventral (B,C) hippocampus produced by an injection in nucleus reuniens (case RE-20). Note the dense concentration of labeled fibers restricted to the stratum lacunosum moleculare of CA1 of the dorsal (A) and ventral (B) hippocam- pus and the molecular layer of the ventral subiculum (C). Note also pronounced labeling in the perirhinal cortex (mainly layer 1) and the (dorsal) lateral entorhinal cortex. For abbreviations see list. Scale bar ⫽ 600 m for A; 1,000 m for B,C. The Journal of Comparative Neurology. DOI 10.1002/cne Fig. 7. Low-magnification darkfield photomicrographs through the ventral hippocampus comparing patterns of labeling in the entorhinal cortex (EC) produced by a caudal (case RE-20; A) and rostral (case aRE-9; B–D) injection in nucleus reuniens. Note the caudal RE injection 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) and in the medial entorhinal cortex (ECm) at three levels of the ventral hippocampus (B–D). Note also the presence of substantial labeling with the rostral RE injection in the ventral subiculum, presubiculum and parasubiculum. For abbreviations see list. Scale bar ⫽ 1,000 m. The Journal of Comparative Neurology. DOI 10.1002/cne 782 Fig. 8. Low-magnification darkfield photomicrographs of transverse sections through the anterior forebrain (A) and ventral hippocampus (B,C) showing patterns of labeling produced by an injection in the perireuniens nucleus (case pRE-1). Note dense labeling in the R.P. VERTES ET AL. medial, ventral, and ventrolateral orbital cortices of the prefrontal cortex (A) as well as in the ventral subiculum and lateral entorhinal cortex at two levels of the ventral hippocampus (B,C). For abbreviations see list. Scale bar ⫽ 750 m for A; 1,000 m for B,C. The Journal of Comparative Neurology. DOI 10.1002/cne EFFERENTS OF REUNIENS AND RHOMBOID NUCLEI 783 Fig. 9. Schematic representation of labeling present in select sections through the forebrain and diencephalon (A–U) and ventral hippocampus (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 of Swanson (1998). For abbreviations see list. Anterior levels of the forebrain (preseptum; Fig. 9A– G). At the anterior forebrain (Fig. 9A–G) labeled fibers were concentrated in medial and ventrolateral regions of the prefrontal/frontal cortex as well as within the dorsal and ventral striatum. Specifically, labeled axons spread widely 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, The Journal of Comparative Neurology. DOI 10.1002/cne 784 R.P. VERTES ET AL. Figure 9 (Continued) The Journal of Comparative Neurology. DOI 10.1002/cne EFFERENTS OF REUNIENS AND RHOMBOID NUCLEI Figure 9 785 (Continued) The Journal of Comparative Neurology. DOI 10.1002/cne 786 PL, and MO (Fig. 10A) but extended uniformly to all layers of AC. Although present, considerably fewer labeled fibers were observed in VO, VLO, anterior piriform cortex, and claustrum (Fig. 9A–D), and only trace amounts were seen laterally in the lateral frontal polar (FPl) and dorsal agranular insular cortices (AId). More caudally in the anterior forebrain (Fig. 9E–G), labeling remained pronounced along the inner wall of mPFC, spreading fairly evenly across all layers of AC and AGm, but much denser in inner (5/6) than outer layers (1–3) of PL and IL (Fig. 9E–G). Similar to RE, labeled fibers in layers 2/3 of AC, PL, and IL were oriented predominantly mediolaterally, parallel to cell layers of these regions. Dense collections of labeled axons were also observed 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 its anterior pole, the entire rostrocaudal extent of ACC was heavily labeled (Fig. 9F–J). Although labeling was stronger in the core than in the shell of ACC, both regions were densely labeled, particularly the core region adjacent to the anterior commissure (see Fig. 10B,C). Although the majority of labeled fibers within the ventrolateral striatum (CP) appeared bound for the anterior forebrain, a significant 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 rostral hippocampus; Fig. 9H–O). At anterior levels of the septum (Fig. 9H–K), labeling was largely confined to dorsomedial and ventrolateral regions of cortex and to the nucleus accumbens/olfactory tubercle. Within the dorsomedial cortex, significant numbers of labeled fibers were present in AGm, capping the cingulum bundle, most heavily in layers 1 and 5/6. The medially adjacent AC was less prominently labeled. On the ventrolateral convexity of cortex (Fig. 9H–K), labeled axons, bordering the external capsule, were found within CLA and to a lesser extent in the endopiriform nucleus (EN). The entire traverse of CLA was strongly labeled (Fig. 9C–O). Lateral to CLA, the insular cortex, extending from AIv to AIp, was moderately labeled. As seen rostrally, labeled fibers blanketed caudal parts of ACC and also spread heavily to the ventrally adjacent OT. Apart from light to moderate labeling in the ventromedial CP (mainly fibers of passage) and the lateral septum, remaining regions of the basal forebrain were sparsely labeled. This included the medial, lateral, and magnocellular preoptic nuclei; diagonal band nuclei; and substantia innominata (SI). More caudally within the basal forebrain (Fig. 9L–O), labeling was largely confined to dorsomedial and ventrolateral regions of cortex. Although significant numbers of labeled axons continued to be present in AGm (all layers) and in CLA, they thinned considerably in AIp. Subcortically, modest numbers of labeled fibers located within the bed nucleus of stria terminalis (BST), SI, and medial regions of basal forebrain/rostral diencephalon were destined mainly for the anterior forebrain (Fig. 9L–O). Posterior levels of the forebrain (anterior hippocampus to the caudal diencephalon; Fig. 9P–U). Similar to rostral 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 negligible elsewhere. Within the cortex, a continuum of labeled fibers of the dorsomedial cortex, beginning rostrally in R.P. VERTES ET AL. AGm/AC, extended caudally to the dorsal retrosplenial cortex (RSCd), lateral/dorsolateral to CB (Fig. 11A). Although present throughout all layers, they were most heavily concentrated in inner layers (5/6) of RSCd. The primary somatosensory cortex (SSI), rostrally, and the posterior parietal cortex (PPC), caudally, lateral to RSCd, were moderately labeled. Some of this labeling, however, as well as that continuous with it within special sensory regions of cortex (Fig. 9R–U), represents fibers bound for the hippocampus/parahippocampus (see below). Labeled fibers continued to occupy ventrolateral regions of cortex, localized to AIp, rostrally (Fig. 9P–R), and to ectorhinal and perirhinal cortices, caudally (Figs. 9S–U, 11B). Labeling was moderate and largely confined to inner layers of AIp, ECT, and PRC. Although fewer than seen with RE injections, moderate numbers of labeled fibers were present in the dorsal hippocampus, forming a tight band in slm of CA1 (Fig. 11A). Subcortically, labeling was virtually restricted to the amygdala. The basolateral and basomedial nuclei were moderately to densely labeled (Figs. 9P–T, 11B), the medial and lateral posterior cortical nuclei lightly labeled. There was a virtual absence of labeling throughout the diencephalon, both in the hypothalamus and in the thalamus. Ventral hippocampal region (Fig. 9AA–II). At caudal levels of the hippocampus (Fig. 9AA–II), labeling was confined to the hippocampal formation (CA1 and parts of the subiculum) and adjacent regions of cortex: RSC, occipital cortex (OC), ECT, PRC, and EC. As with the dorsal hippocampus, a narrow band of labeled fibers was present within the outer molecular layer of the dorsal subiculum/ dorsal CA1 (Fig. 9AA–CC). Unlike that of RE, this labeling did not extend dorsoventrally throughout CA1/ventral subiculum of the rostroventral hippocampus (see Fig. 9AA–CC) but was restricted to dorsal aspects of HF (see Fig. 9AA–CC). This is depicted in the photomicrograph in Figure 12A. More caudally, however, labeled fibers of the dorsal subiculum joined those of the ventral subiculum, to form a continuous strip within the molecular layer of subiculum of the ventral HF (Figs. 9DD–GG, 12,B,C). As with the subiculum, the presubiculum (layer 1) was heavily labeled (Figs. 9GG–II, 12C,E); the para- and postsubiculum were lightly to moderately labeled. In contrast to RE, caudal levels of the retrosplenial cortex were densely labeled, particularly the lateral agranular RSC. As depicted (Fig. 9AA,BB), sizeable numbers of labeled fibers were present dorsal/dorsolateral to the splenium of corpus callosum, mainly within RSCd and RSCagl, and continued to occupy this same position throughout the caudal extent of RSC (Fig. 9AA–II, 12A– C,E). Although fewer than in RSC, significant numbers were also found in the dorsal occipital area (Fig. 12C,E), ventral to the retrosplenial cortex. Some of these, however, appeared bound for ECT, PRC, and ECl (Fig. 9CC– II). Among parahippocampal sites (ECT, PRC, EC), labeling was densest within layers 1, 5/6 of ECT and layers 1 and 4 – 6 of the ventrally adjacent ECl (Figs. 9DD–II, 12A–D). Caudally, the pronounced labeling on the lateral convexity of cortex (Fig. 9GG–II) variously in OC and posterior parietal and ventral temporal cortices appeared mainly terminal. Central medial nucleus For comparisons with injections in RE and RH, control injections were made in the CEM of thalamus, located The Journal of Comparative Neurology. DOI 10.1002/cne Fig. 10. A,B: Low-magnification darkfield photomicrographs of transverse sections through the anterior forebrain showing patterns of labeling in the medial prefrontal cortex (mPFC) and the dorsal and ventral striatum produced by an injection in the rhomboid nucleus (case RH-7). Note dense labeling in inner layers (5/6) of the medial orbital and prelimbic cortices of mPFC (A) as well as in the nucleus accumbens (ACC) and adjacent ventromedial parts of the dorsal striatum. C: High-magnification darkfield photomicrograph from B (arrows) showing substantial numbers of labeled fibers in the core and shell of ACC, particularly in the core of ACC, dorsal and lateral to the anterior commissure, as well as in ventrolateral dorsal striatum. For abbreviations see list. Scale bar ⫽ 750 m for A,B; m 400 m for C. Fig. 11. Low-magnification darkfield photomicrographs of transverse sections through dorsal (A) and ventrolateral (B) regions of the forebrain showing labeling produced by an injection in the rhomboid nucleus (case RH-7). Note dense labeling within stratum lacunosum moleculare of CA1 of the dorsal hippocampus and within several regions of cortex, including the retrosplenial, primary, and secondary motor and primary somatosensory cortices, dorsally (A), as well as within the basal lateral nucleus of the amygdala and parahippocampus (ectorhinal, perirhinal, and entorhinal cortices), ventrally (B). For abbreviations see list. Scale bar ⫽ 800 m for A; 1,000 m for B. The Journal of Comparative Neurology. DOI 10.1002/cne EFFERENTS OF REUNIENS AND RHOMBOID NUCLEI 789 Fig. 12. A–C: Low-magnification darkfield photomicrographs showing patterns of labeling at three levels of the ventral hippocampus produced by an injection in the rhomboid nucleus (case RH-7). Note pronounced labeling in the dorsal subiculum/dorsal CA1 of the rostroventral hippocampus but not in the ventral subiculum/ventral CA1 at this level (A) and labeling in the ventral subiculum at caudal levels 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 and 5 of the ectorhinal and perirhinal cortices. D,E: High-magnification darkfield photomicrographs from C (arrowheads) showing heavy labeling within the ectorhinal cortex (D) and in the occipital and temporal cortices and layer 1 of the postsubiculum (E). For abbreviations see 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 790 R.P. VERTES ET AL. Fig. 13. Low-magnification darkfield photomicrographs of transverse sections through the rostral forebrain showing patterns of labeling produced by an injection in the central medial nucleus of the midline thalamus (case CEM-10). Note dense labeling in dorsal agranular 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. dorsal to RH. Although there was some overlap in patterns of labeling with CEM injections compared with RE/RH injections, differences predominated. Labeling produced by CEM injections was virtually restricted to the rostral forebrain; few labeled fibers were seen caudal to the level of CEM. Within the anterior forebrain, labeling was pronounced throughout FPm (all layers), extending from its anterior tip to its juncture with AGm (Fig. 13A). The laterally adjacent FPl was lightly labeled with rostral CEM injections and moderately labeled with caudal CEM injections. Significant numbers of labeled axons occupied dorsomedial regions of the cortex caudal to FPm within AGm and to a lesser extent in AC (Fig. 13A,B). There was a progressive decline in labeling at successive caudal levels of AGm/ AC, continuing to the retrosplenial cortex, which was virtually devoid of labeled fibers. The rostrocaudal extent of the dorsal agranular insular cortex (AId) was densely labeled (Fig. 13A,B). Considerably fewer labeled fibers were present in the caudally adjacent AIp, with the exception of caudal pole of AIp, bordering the ectorhinal cortex. A restricted strip of labeled fibers stretching across layer 5 of AIp, ECT, and PRC was observed. Subcortically, labeling was heavy within the dorsal and ventral (ACC) striatum, the olfactory tubercle, and the basolateral nu- cleus (BLA) of the amygdala. Massive numbers of labeled fibers were present within ACC (bordering the anterior commissure) as well as within ventrolateral/lateral regions of CP, rostrally, and the entire lateral two-thirds of CP, caudally. This labeling as well as that of the adjacent olfactory tubercle is depicted at two levels of the forebrain in the photomicrographs in Figure 13. Amygdaloid labeling was pronounced within, and was essentially restricted to, anterior and posterior divisions of BLA (Fig. 14A,B). DISCUSSION We examined, compared, and contrasted the efferent projections of nucleus reuniens (RE) and the rhomboid nucleus (RH) of the midline thalamus. Main RE targets are the prefrontal cortex, parahippocampal cortex (ectorhinal, perirhinal, and entorhinal cortices), and hippocampal formation. RH distributes more widely than RE, that is, to most RE projection sites, but also to somatosensory, posterior parietal, retrosplenial, temporal, and occipital cortices as well as to the nucleus accumbens and basolateral nucleus of amygdala. RE and RH projections differ from 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- The Journal of Comparative Neurology. DOI 10.1002/cne EFFERENTS OF REUNIENS AND RHOMBOID NUCLEI 791 sory regions of cortex and none to the hippocampus, projecting instead to the motor cortex and dorsal striatum. There is a shift in patterns of projections from ventral to dorsal regions of the ventral midline thalamus such that main targets: 1) of RE are limbic cortex and hippocampus; 2) of RH are limbic and sensorimotor cortices, hippocampus, ACC, OT, and BMA/BLA; and 3) of CEM are motor cortex, dorsal striatum, and subcortical sites innervated by RH. In effect, the ventral-to-dorsal gradient in projections represents a transition from virtually solely limbic (RE), to sensorimotor/limbic (RH), to largely motor/limbic (CEM). Overall, the ventral midline thalamus is strategically positioned to exert significant control over fairly widespread regions of the cortex (limbic, sensory, motor), the hippocampus, the dorsal and ventral striatum, and the basal nuclei of the amygdala, possibly involved in coordinating limbic and sensorimotor functions. that RE projects substantially to the anterior pole of the PFC (rostral to the genu of CC), that is, to FPm, anterior PL and AC, orbital cortices (MO, VO, VLO), dorsal and ventral agranular insular cortices, and anterior piriform cortex. In accord with the findings of Van der Werf et al. (2002), we showed that the orbital cortex receives particularly dense projections from pRE. Previous studies have described relatively massive RE projections to the ventral hippocampus but at best light ones to the dorsal hippocampus, leading to the conclusion that RE distributes to the ventral but not dorsal HF (Herkenham, 1978; Ohtake and Yamada, 1989; Su and Bentivoglio, 1990; Wouterlood et al., 1990; Dolleman-Van der Weel and Witter, 1996; Risold et al., 1997). For instance, following their findings of modest RE projections to the dorsal hippocampus, Risold et al. (1997) suggested that previous demonstrations of stronger projections (Wouterlood et al., 1990) probably resulted from the spread of injections to the overlying rhomboid nucleus. Although RH distributes to the dorsal hippocampus (Berendse and Groenewegen, 1991, present results), the projections are more restricted and less robust than those from RE. In contrast to our results, some reports (Herkenham, 1978; Ohtake and Yamada, 1989) but not others (Wouterlood et al., 1990; Risold et al., 1997) have described fairly widespread RE projections to several subcortical sites, including the nucleus accumbens, OT, septum, bed nucleus of stria terminalis, medial and lateral preoptic areas, basal nuclei of amygdala, median eminence (ME), lateral hypothalamus, ventral tegmental area, pretectum, superior colliculus, and midbrain central gray. With the exception of projections to the rostral pole of ACC, and some to OT and LS, we failed to observe (terminal) labeling in any of these structures. Wouterlood et al. (1990) similar failed to detect such labeling and argued that earlier descriptions probably resulted from the inclusion of parts of the hypothalamus, particularly the paraventricular nucleus of hypothalamus, in the injections. Consistent with this, we observed moderate, and in some cases fairly dense, labeling in many of the above-mentioned structures with injections that spread ventrally to the paraventricular nucleus or to the posterior nucleus of the hypothalamus (see also Vertes et al., 1995). Although previous reports have shown that RE heavily targets EC, studies differ in their description of precise patterns of innervation of EC, that is, strong projections to ECl and weak ones to ECm (Herkenham, 1978), the reverse (ECm ⬎ ECl; Risold et al., 1997), or essentially equal density (Wouterlood et al, 1990). The present demonstration that the rostral RE preferentially distributes to ECm and the caudal RE to ECl may explain these differences. Summary of RE projections and comparisons with previous studies The primary prefrontal/frontal targets of RE were the medial frontal polar cortex (FPm), the medial and ventral orbital cortices, AGm, AC, PL (anterior and posterior divisions), and IL (of mPFC), and the dorsal, ventral and posterior agranular insular cortices. The main RE targets outside of the prefrontal cortex (PFC) were the rostral retrosplenial cortex and parahippocampus/hippocampus. As described, RE distributes massively to the parahippocampus and HF; that is, to the perirhinal and entorhinal cortices, the slm of CA1 of the dorsal and ventral hippocampus, the molecular layer of the dorsal and ventral subiculum, and the parasubiculum and significantly but somewhat less densely to the ectorhinal (postrhinal) cortex and the pre- and postsubiculum. There was an absence of RE projections to CA2 and CA3 fields of Ammon’s horn and to the dentate gyrus. The rostral RE distributes more heavily to the medial EC than to the lateral EC, whereas the caudal RE (caudal two-thirds of RE) projects more densely to the lateral (dorsal division of ECl) than to the medial EC. Although RE projections to the hippocampus have been well described (Herkenham, 1978; Wouterlood et al., 1990; Wouterlood, 1991; Dolleman-Van der Weel and Witter, 1996; Bokor et al., 2002), few reports have examined overall patterns of RE projections (Herkenham, 1978; Ohtake and Yamada, 1989; Van der Werf et al., 2002). Perhaps the most complete analysis of RE projections was an early study by Herkenham (1978) using the autoradiographic technique. Followup studies, using newer tracers, have concentrated on RE projections to the hippocampus (Wouterlood et al., 1990) and to the EC (Wouterlood, 1991) or projections from specific regions of RE (Risold et al., 1997). In general, the findings of previous studies support the present results. With some exceptions, there appears to be agreement that RE targets mainly “limbic” (neo/allo) cortex and the hippocampus and to a considerably lesser degree subcortical structures. In partial contrast to earlier reports, however, we observed considerably stronger projections to commonly labeled regions (particularly PFC, dorsal hippocampus, and ECl) and projections to a number of sites not previously described. Consistent with earlier findings, we showed that RE distributes significantly to the 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 Summary of RH projections and comparisons with previous studies The major cortical projection sites of RH are the FPm, medial orbital cortex, mPFC, retrosplenial cortex, posterior parietal cortex, occipital cortex, parahippocampus, and HF. The main subcortical sites are the claustrum, ACC, OT, LS, and basal nuclei of the amygdala (BLA and BMA). Similar to RE, RH fibers distribute widely over the PFC/frontal cortex, terminating heavily in FPm, medial orbital cortex, AGm, AC, PL, and IL (of mPFC) and moderately in the lateral frontal polar, the anterior piriform, Fig. 14. Low-magnification darkfield photomicrographs of transverse sections through the forebrain showing patterns of labeling in the 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 of amygdala at these levels. For abbreviations see list. Scale bar ⫽ 500 m. The Journal of Comparative Neurology. DOI 10.1002/cne EFFERENTS OF REUNIENS AND RHOMBOID NUCLEI 793 ventral orbital, and insular (AId, AIv, AIp) cortices. Additional cortical projection sites include caudal parts of AGm and AC and the retrosplenial, somatosensory, posterior parietal, temporal, and occipital cortices. Although less pronounced than RE, RH also distributes significantly to the hippocampus and parahippocampus; that is, to slm of CA1 of the dorsal and ventral hippocampus, the molecular layer of the dorsal subiculum, the posteroventral subiculum, and the postsubiculum as well as to the perirhinal cortex (flanking RF) and inner layers (5/6) of the dorsal ECl. Few reports have examined the efferent projections of RH (Ohtake and Yamada, 1989; Berendse and Groenewegen, 1991; Van der Werf et al., 2002). In general, the present findings are consistent with earlier demonstrations of a widespread distribution of RH fibers to the cortex and parts of the striatum. For instance, with regard to the cortex, Van der Werf et al. (2002) noted that, “in contrast to the other intralaminar and midline nuclei, the projections of Rh are not confined to limbic structures and associated cortices, but reach primary and secondary motor and sensory cortices as well.” It is nonetheless clear, however, that RH distributes less densely to sensorimotor regions of cortex than to various “limbic” regions of cortex, including the hippocampus, suggesting that RH, like RE, exerts primary influence over subcortical/cortical limbic structures. Although previous reports have described RH projections to HF (Yanaghihara et al., 1987; Su and Bentivoglio, 1990; Berendse and Groenewegen, 1991; Van der Werf et al. 2002), they appear to be considerably less pronounced than shown here. For instance, Berendse and Groenewegen (1991) described relatively prominent RH projections to the dorsal subiculum and dorsal CA1 of the ventral hippocampus but failed to confirm our demonstration of projections to CA1 of the dorsal hippocampus and to the posteroventral subiculum. This may involve differing locations of RH injections. Consistent with the present results, Ohtake and Yamada (1989) described prominent RH projections to ACC, spreading virtually throughout ACC, whereas Berendse and Groenewegen (1991) reported that RH distributes fairly selectively to the ventrolateral shell of ACC. Although RH fibers are more densely concentrated in the core than in the shell of ACC (present results), the findings of several studies with retrograde tracers (Ohtake and Yamada, 1989; Su and Bentivoglio, 1990; Brog et al., 1993; Otake and Nakamura, 1998), showing labeled cells in RH with injections throughout ACC, support a rather widespread distribution of RH efferents to ACC. In contrast to the present and earlier findings (Berendse and Groenewegen, 1991) that RH projects to limited sites of the diencephalon and basal forebrain, Ohtake and Yamada (1989) reported that RH distributes terminally to several subcortical structures, including the BST; the lateral, medial, basomedial, and basolateral nuclei of the amygdala; the anterior and lateral hypothalamus; the zona incerta; and the ventrolateral, ventromedial, ventroposterior, central medial and submedial nuclei of thalamus. With the exception of RH projections to BLA and BMA, we essentially failed to observe labeling in these structures (see also Berendse and Groenewegen, 1991). The injections of Ohtake and Yamada (1989) were large and seemed to extend beyond the boundaries of RH, rais- ing the possibility that their labeling originated from thalamic groups outside of RH. Functional significance Nucleus reuniens. We showed that the output of RE is directed to the hippocampus, parahippocampus, and various regions of the prefrontal cortex. These “limbic” structures of cortex serve a well-recognized role in various forms of memory processing (Baddeley, 1986, 1998; Dudai, 1989; Goldman-Rakic, 1994, 1995; Eichenbaum et al., 1996; Eichenbaum and Cohen, 2001; Fuster, 2001; Vertes et al., 2004a; Vertes, 2005). With the possible exception of RH, no other nucleus of the thalamus displays a similar pattern of projections. Hence, RE is uniquely positioned to influence simultaneously major structures of the brain (HF and mPFC) subserving memory. Although the output of RE is relatively restricted, RE receives a diverse and widely distributed set of afferent projections, mainly from limbic and limbic-associated structures of the cortex, basal forebrain, hypothalamus, amygdala, and brainstem (Herkenham, 1978; Risold et al., 1997; Canteras and Goto, 1999; Krout et al., 2002; OluchaBordonau et al., 2003; McKenna and Vertes, 2004). This suggests that RE is a critical site for the convergence of a diverse array of limbic/limbic related information and its subsequent transfer to its main targets, the hippocampus/ parahippocampus and the orbitomedial PFC. In previous examinations of the efferent projections of the mPFC in rats (Vertes, 2002, 2004), we showed that all four divisions of mPFC (IL, PL, AC, and AGm) project massively to RE. Aside from RE, only the mediodorsal nucleus of thalamus receives similar projections (Groenewegen, 1988; Sesack et al., 1989; Hurley et al., 1991; Price, 1995; Öngür and Price, 2000; Vertes, 2002; Gabbott et al., 2005). Several reports for various species have demonstrated pronounced projections from the hippocampus to the mPFC (Swanson, 1981; Irle and Markowitsch, 1982; Cavada et al., 1983; Goldman-Rakic et al., 1984; Ferino et al., 1987; Jay et al., 1989; van Groen and Wyss, 1990; Jay and Witter, 1991; Carr and Sesack, 1996), but there are no return 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; Takagishi and Chiba, 1991; Buchanan et al., 1994). For instance, Laroche et al. (2000) recently noted that “unlike other neocortical areas such as the perirhinal or entorhinal cortices, 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 prefrontal cortex in the rat.” The demonstration, then, of pronounced mPFC projections to RE and similar dense RE projections to the hippocampus and EC, coupled with the absence of mPFC to HF/EC projections, suggests that RE represents a critical relay in the transfer of information from the mPFC to the HF/EC. All cortical areas receive and send projections to the thalamus (Jones, 1985). The conventional view is that the cortical output to the thalamus primarily serves to modulate return thalamocortical projections. Although, in part, this is undoubtedly true, the paradoxical findings of greater than tenfold more corticothalamic than thalamocortical fibers suggests that cortical projections to thalamus do not merely modulate return projections to cortex. In this regard, Llinas et al. (1998) proposed that the thal- The Journal of Comparative Neurology. DOI 10.1002/cne 794 amus may serve as a way station for intracortical communication. Specifically, they suggested that the thalamus should not be viewed simply as a gateway to the cortex; rather, “the thalamus represents a hub from which any site in the cortex can communicate with any other such site or sites.” Along these lines, we would suggest that the midline thalamus (or RE and RH) might serve critically to interconnect select cortical and subcortical limbic structures, that is, an important conduit (hub) in the transfer of information among these structures. In contrast to direct connections between these structures (Groenewegen et al., 1990; Gabbott et al., 2003), information flow between them, by way of RE, would appear subject to modulatory influences acting on RE (McKenna and Vertes, 2004) that may serve to “gate” the transfer of information. In effect, depending on the type (or mode) of input it receives, RE may differentially channel information between specific sets (or subsets) of forebrain structures appropriate to the demands of the behavioral situation, in a sense act as a master switch. Rhomboid nucleus. Similar to RE, RH may also serve a critical role in intracortical and cortical-subcortical communication, but for a more diverse set of structures than for RE. In this regard, RH and RE receive similar sets of afferents from the brainstem and hypothalamus but largely different inputs from the forebrain. Specifically, reflecting its output, RH receives afferents from the limbic forebrain, but additionally pronounced projections from primary and secondary motor cortices (AGl and AGm) and the primary somatosensory cortex (Vertes et al., 2004b; Owens, 2005). Based on its inputs and outputs, RH may bridge sensorimotor and limbic domains, possibly providing 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 of thalamus have been designated “nonspecific thalamus,” differentiating them from relay nuclei of the thalamus. As discussed, the early notion that midline nuclei of thalamus distribute widely throughout the cortical mantle and hence exert global effects on the cortex has been revised by findings (Berendse and Groenewegen, 1991; Van der Werf et al., 2002; present results) showing that each of the midline nuclei exhibits a unique and restricted set of cortical (and subcortical) projections. With regard to these projections, we described a ventral-to-dorsal gradient in projections of the ventral midline thalamus from almost entirely “limbic” (RE) to a progressively greater somatic/ limbic mix (RH/CEM). The ventral midline thalamus is ideally positioned to relay diverse, mainly limbic, information from various sources to forebrain structures involved in emotional/cognitive and sensorimotor aspects of behavior. Physiological effects of the ventral midline thalamus on the hippocampus and mPFC. As described, major RE targets are the hippocampus, EC, and mPFC. It has recently been shown stimulation of RE/RH produces strong excitatory actions at CA1 of the hippocampus (Dolleman-Van der Weel et al., 1997; Bertram and Zhang 1999), 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 RE stimulation produced 1) large negative-going field potentials at stratum lacunosum-moleculare of CA1 indicative R.P. VERTES ET AL. of a pronounced depolarizing action of RE on distal apical dendrites of CA1 pyramidal cells and 2) a marked pairedpulse facilitation of evoked potentials at CA1. They proposed that RE may “exert a persistent influence on the state of pyramidal cell excitability,” depolarizing cells to close to threshold for activation by other excitatory inputs. Consistent with this, Bertram and Zhang (1999) compared the effects of RE (midline thalamic) and CA3 stimulation on various population measures at CA1 and showed that RE actions on CA1 were equivalent to, and in some cases considerably greater than, those of CA3 on CA1. They concluded that the RE projection to the hippocampus “allows for the direct and powerful excitation of the CA1 region. This thalamohippocampal connection bypasses the trisynaptic/commissural pathway that has been thought to be the exclusive excitatory drive to CA1.” More recently, Zhang and Bertram (2002) similar demonstrated that RE stimulation produced short-term (evoked responses) and long-term (paired-pulse facilitation and LTP) excitatory effects at EC and concluded that RE “plays a significant role in limbic physiology and may serve to synchronize activity in this system.” Finally, we recently showed (Viana Di Prisco and Vertes, 2006) that stimulation of the ventral midline thalamus (RE/RH) produced 1) large-amplitude, monosynaptically elicited evoked potentials dorsoventrally throughout the mPFC, with the largest effects elicited at the infralimbic (IL) and prelimbic (PL) cortices, and 2) pronounced paired-pulse facilitation at IL and PL. In summary, RE/RH target predominantly limbic forebrain structures and exert pronounced excitatory actions on them. 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