Projections of the central medial nucleus of the thalamus in the rat: Node in cortical, striatal and limbic forebrain circuitry

Neuroscience 219 (2012) 120–136

PROJECTIONS OF THE CENTRAL MEDIAL NUCLEUS OF THE THALAMUS IN THE RAT: NODE IN CORTICAL, STRIATAL AND LIMBIC FOREBRAIN CIRCUITRY R. P. VERTES, a* W. B. HOOVER a AND J. J. RODRIGUEZ b,c,d

Abstract—The central medial nucleus (CM) of thalamus is a prominent cell group of the rostral intralaminar complex of the thalamus. No previous report in the rat has comprehensively described the projections of CM. Using the anterograde anatomical tracer, Phaseolus vulgaris leucoagglutinin, we examined the efferent projections of CM, comparing projections from rostral (CMr) and caudal (CMc) regions of CM. We showed that the central medial nucleus distributes substantially to several cortical sites and to a limited number of subcortical structures. The primary CM targets were anterior and posterior regions of cortex, the claustrum, the caudate-putamen, the nucleus accumbens (ACC), the olfactory tubercle, and the amygdala. CMr and CMc distribute to several of the same structures but essentially to different parts of these structures. By comparison, CMr more strongly targets limbic structures, CMc more heavily sensorimotor structures. Main CMr projection sites were the medial agranular, anterior cingulate, prelimbic, dorsolateral orbital and dorsal agranular insular cortices, the dorsal striatum, the ACC, and the basolateral nucleus of the amygdala. Main CMc cortical projection sites were the ventrolateral, lateral and dorsolateral orbital cortices, dorsal, ventral and posterior agranular insular cortices, visceral cortex, primary somatosensory and motor cortices, and perirhinal cortex. Main CMc subcortical projection sites were the dorsal striatum and the lateral, central, anterior cortical, and basomedial nuclei of amygdala. The largely complementary output of CMr and CMc to diverse areas of cortex and to regions of the striatum and amygdala suggest a combined CM influence over a widespread area of the anterior cortex and throughout the dorsal and ventral striatum and the amygdala. CM projections to limbic and sensorimotor structures of the rostral forebrain suggest that CM may serve to integrate affective, cognitive and sensorimotor functions for goal-directed behavior. Ó 2012 Published by Elsevier Ltd. on behalf of IBRO.

a

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

b

IKERBASQUE, Basque Foundation for Science, 48011 Bilbao, Spain

c Department of Neurosciences, University of the Basque Country UPV/EHU, 48940 Leioa, Spain d

Institute of Experimental Medicine, ASCR, Videnska 1083, 142 20 Prague, Czech Republic

*Corresponding author. Tel: +1-561-297-2362; fax: +1-561-2972363. E-mail address: [email protected] (R. P. Vertes). Abbreviations: AAA, anterior amygdaloid area; ac, anterior commissure; AC, anterior cingulate cortex; ACC, nucleus accumbens; AGm, medial agranular (frontal) cortex; AGl, lateral agranular (frontal) cortex; AH, anterior nucleus of hypothalamus; AI,d,p,v, agranular insular cortex, dorsal, posterior and ventral divisions; AM, anteromedial nucleus of thalamus; AON, anterior olfactory nucleus; AUD, auditory cortex; AV, anteroventral nucleus of thalamus; BLA, basolateral nucleus of amygdala; BMA, basomedial nucleus of amygdala; BST, bed nucleus of stria terminalis; CEA, central nucleus of amygdala; CL, central lateral nucleus of thalamus; CLA, claustrum; CM,c,r, central medial nucleus of thalamus, caudal division, rostral division; COA,a,p, cortical nucleus of amygdala, anterior, posterior division; C-P, caudate-putamen, striatum; DLO, dorsolateral orbital cortex; DM, delayed matching to sample/position task; DMh, dorsomedial nucleus of hypothalamus; DNM, delayed non-matching to sample/position task; EC, entorhinal cortex; ECT, ectorhinal cortex; EP, endopiriform nucleus; GP, globus pallidus; GU, gustatory cortex; IAM, interanteromedial nucleus of thalamus; IL, infralimbic cortex; ILt, intralaminar nuclei of thalamus; IMD, intermediodorsal nucleus of thalamus; LA, lateral nucleus of amygdala; LD, lateral dorsal nucleus of thalamus; LH, lateral habenula; LHy, lateral hypothalamus; LO, lateral orbital cortex; LP, lateral posterior nucleus of thalamus; LPO, lateral preoptic area; LS, lateral septum; MA, magnocellular preoptic nucleus; MD, mediodorsal nucleus of thalamus; MO, medial orbital cortex; mPFC, medial prefrontal cortex; MPO, medial preoptic area; MS, medial septum; mt, mammillothalamic tract; OT, olfactory tubercle; PB, phosphate buffer; PC, paracentral nucleus of thalamus; PFC, prefrontal cortex; PH, posterior hypothalamus; PHA-L, Phaseolus vulgaris-leucoagglutinin; PIR, piriform cortex; PL, prelimbic cortex; PO, posterior nucleus of thalamus; PRC, perirhinal cortex; PT, paratenial nucleus of thalamus; PTL, posterior parietal cortex; PV, paraventricular nucleus of thalamus; RE, nucleus reuniens of thalamus; RF, reticular formation; RH, rhomboid nucleus of thalamus; RSC, retrosplenial cortex; RT, reticular nucleus of thalamus; SF, septofimbrial nucleus; SI, substantia innominata; SMT, submedial nucleus of thalamus; SSI, primary somatosensory cortex; SSII, secondary somatosensory cortex; TBS, Tris-buffered saline; TEA, temporal association area; TR, postpiriform transition area; VAL, ventral anterior-lateral nucleus of thalamus; VB, ventral basal nucleus of thalamus; VISC, visceral cortex; VLO, ventrolateral orbital cortex; VM, ventral medial nucleus of thalamus; VMh, ventromedial nucleus of hypothalamus; VO, ventral orbital cortex; ZI, zona incerta; 3V, third ventricle.

Key words: medial prefrontal cortex, insular cortex, nucleus accumbens, striatum, basolateral nucleus of amygdala, working memory, limbic thalamus.

INTRODUCTION The central medial nucleus (CM) is a prominent cell group of the rostral intralaminar complex of the thalamus which also includes the paracentral (PC) and central lateral nuclei. CM extends rostrocaudally over a considerable length of the thalamus (Swanson, 2004). The midline and intralaminar (ILt) nuclei receive a diverse and widespread set of afferent projections (Krout

0306-4522/12 $36.00 Ó 2012 Published by Elsevier Ltd. on behalf of IBRO. http://dx.doi.org/10.1016/j.neuroscience.2012.04.067 120

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et al., 2002; Van der Werf et al., 2002; Vertes, 2002; McKenna and Vertes, 2004). Specifically, CM receives a relatively vast array of projections, primarily from the brainstem and caudal diencephalon. Significant among them are afferents from the brainstem reticular formation (RF). Several early reports in the rat demonstrated that the intralaminar nuclei, together with the zona incerta, were the major forebrain targets of RF projections (Jones and Yang, 1985; Vertes et al., 1986; Vertes and Martin, 1988). These connections constitute a principal component of the ascending RF activating system – or a reticular-ILt-cortical circuit responsible for behavioral and cortical EEG arousal of waking and REM sleep (Steriade and Glenn, 1982; Glenn and Steriade, 1982; Steriade et al., 1982; Kinomura et al., 1996). Damage to this system profoundly alters states of consciousness (Castaigne et al., 1981; Mair, 1994; Schiff, 2008), leading to the view that the midline/ ILt thalamus (including CM) is critically involved in processes of arousal and attention (Paus, 2000; Van der Werf et al., 2002; Schiff, 2008). Whereas CM receives widespread afferents, the output of CM is relatively restricted and mainly directed to rostral regions of the cortex, or to the orbitomedial prefrontal cortex (PFC) (Berendse and Groenewegen, 1991; Van der Werf et al., 2002). This suggests a role for CM in PFC functions such as decision-making and goal-directed behaviors (Dalley et al., 2004; Kolb et al., 2004; Sul et al., 2010). Specifically, CM/ILt lesions have been shown to produce impairments in working memory as assessed by delayed matching (DM) and non-matching (DNM) to sample/position tasks in rats (Burk and Mair, 1998; Mair et al., 1998; Bailey and Mair, 2005; Newman and Burk, 2005; Mitchell and Dalrymple-Alford, 2005, 2006; Mair and Hembrook, 2008). For instance, Mair and Hembrook (2008) demonstrated that pharmacologically or electrically elicited activation of ILt improved performance on a DM to position task, while ILt suppression impaired performance on the task. On the human level, Van der Werf et al. (1999) described severe impairments in declarative memory in a patient with damage to the right side of the intralaminar thalamus which were attributed to ‘‘impaired use of mental flexibility’’. They postulated that this dysfunction ‘‘arose from the loss of cortical activation, caused by deafferentation of the prefrontal cortex through damage to the intralaminar nuclei’’ (Van der Werf et al., 2003). The foregoing indicates, then, a critical role for CM in arousal/attention and cognition and hence the need to understand the pattern of CM projections. While earlier reports in rats have described CM projections to some structures (Berendse and Groenewegen, 1990, 1991; Van der Werf et al., 2002), no previous study has comprehensively examined the output of CM. The present report, then, sought to fully describe the pattern of CM projections throughout the brain.

EXPERIMENTAL PROCEDURES Single injections of Phaseolus vulgaris-leuccoagglutinin (PHA-L) were made into the rostral central medial (CMr) or caudal central medial (CMc) nucleus of the intralaminar thalamus in 42 male

Sprague–Dawley rats (Harlan, Indianapolis, IN) weighing 300– 420 g. The experiments were approved by the Florida Atlantic University IACUC committee and conform to all Federal regulations and National Institutes of Health Guidelines for the care and use of laboratory animals.

PHA-L procedures Powdered lectin from PHA-L was reconstituted to 4–5% in 0.05 M sodium phosphate buffer (PB), 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 40–50 lm. Rats were anesthetized for surgery using an 80 mg/ kg dose of Ketamine (100 mg/ml) and 10 mg/kg dose of Xylazine (20 mg/ml). Positive direct current (7–12 lA) was applied through a Grass stimulator (Model 88) coupled with a high-voltage stimulator (Frederick Haer Co., Bowdoin ME) at 2-s ‘‘on’’/2-s ‘‘off’’ intervals for 40–50 min. After a survival time of 7–10 days, rats were deeply anesthetized with Ketamine/Xylazine (150 mg/kg and 50 mg/kg, respectively), and perfused transcardially with a heparinized buffered saline wash (50–75 ml/animal) followed by a fixative (4% paraformaldehyde, 0.2–0.5% glutaraldehyde in 0.1 M PB, pH 7.4) (300–500 ml/animal). The brains were removed and postfixed overnight in 4% paraformaldehyde 0.1 M PB at 4 °C, pH 7.4. Following postfixing, the brains were transferred to 30% sucrose in 0.1 M PB solution for 2 days at 4 °C. Following sucrose cryoprotection, 50-lm frozen sections were collected in phosphate-buffered saline (PBS, 0.9% sodium chloride in 0.01 M sodium phosphate buffer, pH 7.4) using a freezing microtome. Six series of sections were taken. A complete series of sections was treated with 1% sodium borohydride in 0.1 M PB for 30 min to remove excess reactive aldehydes. Following this, sections were thoroughly rinsed in 0.1 M PB, and then incubated for 60 min at room temperature in 0.5% bovine serum albumin in Tris-buffered saline (TBS) to minimize nonspecific labeling. The sections were then incubated in the primary antiserum directed against PHA-L [biotinylated goat (IgG) anti-PHA-L, Vector Labs, Burlingame, CA] and diluent (0.1% bovine serum albumin in TBS containing 0.25% Triton X-100) at a concentration 1:500 overnight. Sections were then washed in 0.1 M PB (6  4 min) and placed in a 1:500 concentration of biotinylated rabbit antigoat immunoglobulin (IgG) and diluent for 2 h. Sections were washed and then incubated in a 1:100 concentration of peroxidase–avidin complex from the Elite kit (Vector Labs) and diluent for 1 hour. Following another 0.1 M PB wash, the peroxidase reaction product was visualized by incubation in a solution containing 0.022% 3,30 diaminobenzidine (DAB, Aldrich, Milwaukee, WI) and 0.003% H2O2 in TBS for 6 min. Sections were then rinsed again in 0.1 M PB (3  1 min) and mounted onto chrome–alum gelatin-coated slides. An adjacent series of sections from each rat was stained with cresyl violet for anatomical reference. Sections were examined using light and darkfield optics. Injection sites, cells and labeled fibers were plotted on representative schematic coronal sections through the brain using sections adapted from the rat atlas of Swanson (2004). Brightfield and darkfield photomicrographs of injection sites and labeled fibers were taken with a QImaging (Q ICAM) camera mounted on a Nikon Eclipse E600 microscope. Digital images were captured and reconstructed using Nikon Elements 3.0 imaging software (Melville, NY), and adjusted for brightness and contrast using Adobe PhotoShop 7.0 (Mountain View, CA). Sections that were reacted without either the primary or secondary antibodies did not show immunoreactivity (data not shown).

RESULTS The patterns of distribution of labeled fibers throughout the brain following PHA-L injections in the rostral (CMr) and the caudal (CMc) central medial nucleus of the rostral ILt are described. Fig. 1 depicts the sites of injection in

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CMr (Fig. 1A, B) and CMc (Fig. 1C, D) for the schematically illustrated cases (cases 38 and 32). The patterns of labeling obtained with these cases are representative of those observed with non-illustrated cases. Central medial nucleus, rostral part (CMr) (case 38) Fig. 2 depicts the pattern of distribution of labeled fibers throughout the brain following a PHA-L injection in the rostral part of CM (Fig. 1A, B and Fig. 2P). Labeled fibers coursed forward from the site of injection (Fig. 2P) to the anterior forebrain, spreading widely over the cortex (Fig. 2A–D). Labeled axons stretched mediolaterally across the prefrontal cortex most densely concentrated: (1) dorsally/dorsomedially in the medial agranular (AGm) (secondary motor) and the prelimbic (PL) cortices, rostrally (Fig. 2A, B), and in AGm and the anterior cingulate cortex (AC) caudally (Fig. 2C, D); and (2) ventrolaterally in the dorsolateral orbital cortex (DLO), rostrally (Fig. 2A, B) and in the dorsal agranular insular cortex (AId), caudally (Fig. 2C, D). Of these sites, labeling was heaviest in AC and AId (Fig. 2A–D). Some labeled fibers were also present in the lateral agranular cortex (AGl) (primary motor) and in medial orbital cortex (MO), ventral to PL (Fig. 2A–D). There was an absence of labeling in olfactory structures: anterior olfactory nucleus, tenia tecta and anterior piriform cortex (Fig. 2A–D). Further caudally in the anterior forebrain (Fig. 2E–G), labeled fibers of the cortex were largely restricted to AGm

and AC, dorsomedially, and to AId, ventrolaterally. Labeled axons spread to all layers of AGm and AC, most densely concentrated in layers 1, 3 and 6 (Fig. 2E, F). Additional moderate to heavily labeled sites were the nucleus accumbens (ACC), olfactory tubercle (OT), claustrum (CLA), and rostral pole of the dorsal striatum (or caudate-putamen, C-P) (Fig. 2E–G). These patterns of labeling (Fig. 2A–G) are depicted in the micrographs of Fig. 3A–C. As shown, there were three main clusters of fibers: dorsomedially in AGm and AC; laterally in AId and CLA; and ventromedially in ACC and OT with extensions dorsally into C-P. The anterior, core, and shell regions of ACC bordering the anterior commissure were densely labeled (Fig. 3B, C). Light to moderate numbers of labeled axons were also seen in the infralimbic (IL) and PL cortices, mainly confined to deep layers (Fig. 2E, F). Caudally within the anterior forebrain (septal levels) (Fig. 2H–M), labeled fibers spread heavily throughout the dorsal striatum, mainly localized to central regions of C-P, rostrally (Fig. 2H–J) and to dorsomedial aspects of C-P, caudally (Fig. 2K–M). Fig. 4 depicts a dense column of labeled fibers stretching dorsoventrally over the central C-P, rostrally (Fig. 4A, B), and occupying most of the dorsal/dorsomedial sector of C-P, caudally (Fig. 4C, D). While labeled fibers continued to be present in the core of ACC and some in the shell (Fig. 2H–J,), their numbers declined from those of rostral levels. Outside of the dorsal and ventral striatum, light to moderate numbers of labeled

Fig. 1. Low (A, C) and high (B, D) magnification bright-field photomicrographs of transverse sections through diencephalon showing locations of PHA-L injections in the rostral (A, B) and caudal (C, D) central medial (CM) nucleus of the thalamus. Scale bar for A = 1100 lm; for B = 350 lm; for C = 1150 lm; for D = 375 lm. See list for abbreviations.

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Fig. 2. Schematic depiction of labeling present in representative sections through the forebrain (A–S) produced by a PHA-L injection (red dots in P) in the rostral part of the central medial nucleus of thalamus (case 38). Sections modified from the rat atlas of Swanson (2004). See list for abbreviations. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2 (continued)

axons were observed in AGm, AId, CLA and OT. At best minor labeling was present throughout remaining regions

of the basal forebrain including the medial and lateral preoptic areas (Fig. 2J–M).

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Fig. 2 (continued)

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At the rostral diencephalon (Fig. 2N–P), labeled axons were virtually confined to C-P and to the basolateral nucleus (BLA) of the amygdala. As observed rostrally (Fig. 2K–M), labeled fibers of the striatum were localized to the dorsal half of C-P, bordering the lateral ventricle (Fig. 2N–P). The rostro-caudal extent of BLA was heavily labeled; few fibers were present within other parts of the amygdala (Fig. 2N–S). This is exemplified in Fig. 5 showing dense concentrations of labeled axons rostrally (Fig. 5A) and caudally (Fig. 5B) in BLA. Labeling was light outside C-P and BLA, mainly localized to AGm and to the retrosplenial cortex (RSC), dorsomedially (Fig. 2N–R), and to areas bordering the rhinal fissure, ventrolaterally. The latter included the posterior agranular insular cortex (AIp) (Fig. 2N, O) and the perirhinal cortex (PRC) (Fig. 2P–S). At middle to caudal levels of the diencephalon (Fig. 2Q–S), labeling weakened significantly and was essentially restricted to caudal BLA and to parts of the cortex. As rostrally, labeled fibers spread quite heavily throughout BLA, and the relatively few present in the cortex were primarily localized to RSC and perirhinal cortices. Central medial nucleus, caudal part (CMc) (case 32)

Fig. 3. Low-power bright-field photomicrographs of three rostrocaudally aligned (A–C) transverse sections through the rostral forebrain showing patterns of labeling produced by a PHA-L injection in the rostral part of the central medial nucleus. Note the dense labeling: (a) in the medial prefrontal cortex (mPFC) predominantly localized to the medial agranular and anterior cortices (A–C) with some extension ventrally into the prelimbic cortex (A); (b) in the dorsal agranular insular cortex (A–C); (c) in the dorsal striatum (C); and (d) in the nucleus accumbens and the ventrally adjacent OT (B,C). Scale bar for A = 950 lm; for B = 975 lm; for C = 1120 lm. See list for abbreviations.

Fig. 6 depicts the pattern of distribution of labeled fibers throughout the brain following a PHA-L injection in the caudal part of CM (Fig. 1C, D and Fig. 6Q, R). As shown, labeled fibers exiting from the site of injection (Fig. 6Q, R) coursed rostrally to the anterior forebrain mainly destined for lateral regions of the cortex (Fig. 6A–D). At the rostral pole of the cortex (Fig. 6A), labeled axons stretched mediolaterally across the cortex primarily localized to the prelimbic cortex (PL), medially and to parts of the orbital and motor (AGl) cortices, laterally. As depicted schematically (Fig. 6A) and in the micrograph of Fig. 7A, a relatively narrow band of labeled fibers extended laterally from the midline through all layers of PL, continued through deep layers of the ventrolateral (VLO), lateral (LO) and dorsolateral (DLO) orbital cortices, and ended laterally in superficial layers of LO and DLO. Labeling was dense in layers 1 and 3 of DLO. In addition, moderate numbers of labeled fibers were present in AGl, but very few in AGm. Further caudally in the anterior PFC (Fig. 6B–D), labeling was predominantly confined to middle to lateral parts of the cortex, with modest labeling of the medial prefrontal cortex (mPFC), including the IL, PL and AC cortices. As shown, a dense collection of dorsomedially to ventrolaterally oriented fibers occupied the lateral twothirds of the PFC (Fig. 6B–D), localized medially to deep layers of VLO, LO and AGl and to CLA, and laterally to AId and to the dorsally adjacent gustatory (GU) and primary somatosensory (SSI) cortices. These patterns are depicted in the micrographs of Fig. 7B, C showing heavy labeling of layers 1 and 3 of AId (Fig. 7B, C) and moderate labeling of the CLA (Fig. 7C). Labeled fibers were also largely restricted to lateral parts of the cortex caudally in the anterior forebrain (Fig. 6E–G). As depicted, labeled axons lined the lateral

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Fig. 4. Low-power bright-field photomicrographs of four rostrocaudally aligned (A–D) transverse sections through the rostral forebrain showing patterns of labeling in the dorsal striatum (C-P) produced by a PHA-L injection in the rostral part of the central medial nucleus. Note the very dense labeling spread throughout C-P, concentrated rostrally (A,B) dorsoventrally throughout the mid-central C-P, and caudally (C,D) within the medial/ dorsomedial half of C-P. Scale bar for A = 975 lm; for B, C = 1000 lm; for D = 950 lm. See list for abbreviations.

edge of the corpus callosum (anterior forceps) (Fig. 6E,F) and extended from there to ventrolateral regions of cortex, heavily concentrated in superficial layers (1 and 3) of AId and GU and less so dorsally in SSI. Additional moderately labeled sites were the CLA (Fig. 6E–G) and AGl (Fig. 6E, F). Few labeled fibers were present in mPFC or within anterior regions of C-P, ACC and OT. Labeling further caudally in the forebrain (septal levels) (Fig. 6H–K) was restricted to a few sites, the most prominent being ventral/ventromedial parts of the dorsal striatum. As shown schematically (Fig. 6H–K) and in the micrographs of Fig. 7D, E dense aggregates of labeled axons were present rostrocaudally throughout the ventral C-P, lateral/dorsolateral to ACC, rostrally (Fig. 7D), and on the lateral border of the globus pallidus (GP), caudally (Fig. 7E). Relatively significant numbers of fibers were

also present along the lateral convexity of the cortex extending continuously from AC to regions bordering the rhinal fissure, including AId and GU, rostrally (Fig. 6H) and AIp, caudally (Fig. 6I–K). Of these sites, labeling was fairly pronounced in AId, GU and AIp, but less dense in AGl and the somatosensory cortices (SSI and SSII). Additional light to moderately labeled sites were CLA, ACC, OT, and the substantia innominata (SI) (Fig. 6H–K). At the rostral diencephalon (Fig. 6L–Q), labeled axons were virtually confined to the ventrolateral quadrant of the brain: within C-P, AIp, PRC, and the amygdala. Few fibers were visible throughout remaining regions of the cortex or within the thalamus and hypothalamus. The labeled axons of the thalamus largely represented fibers exiting the thalamus from the site of injection (Fig. 6Q, R). As rostrally, labeled fibers of the striatum were mainly found

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Fig. 5. (A, B) Darkfield photomicrographs of transverse sections through the rostral (A) and caudal (B) amygdala showing patterns of labeling produced by a PHA-L injection in the rostral part of the central medial nucleus. Note dense labeling restricted to the basal lateral nucleus of amygdala. C–E: Darkfield photomicrographs of three rostral to caudal transverse sections (C–E) through the amygdala showing patterns of labeling produced by a PHA-L injection in the caudal part of the central medial nucleus. Note pronounced labeling in the central, lateral and basolateral nuclei of amygdala. Scale bar for A = 330 lm; for B = 310 lm; for C–E = 500 lm. See list for abbreviations.

ventromedially in C-P, bordering GP (Fig. 6L, M), with few extending to caudal aspects of C-P (Fig. 6N–R). Striatal labeling was very dense in a region dorsal to the anterior nuclei of the amygdala (Fig. 6L). Of the areas around the rhinal fissure, labeling was heaviest in AIp (particularly layers 3 and 5/6), in visceral cortex (VISC) (Fig. 6L, M) and in the PRC (Fig. 6O–R), and much less pronounced in the ectorhinal (ECT) and piriform cortices (Fig. 6O– Q). Unlike rostral CM injections (see above) wherein amygdala labeling was virtually confined to the basolateral nucleus of amygdala, caudal CM injections gave rise to labeling of several nuclei of the amygdala (Fig. 5C–E). As depicted (Fig. 5L–P), these included parts of the anterior (AAA), central (CEA), medial, anterior cortical (COAa), lateral (LA), basolateral and basomedial (BMA) nuclei of amygdala. Labeling was densest in LA (Fig. 5C, D), lateral aspects of BLA, BMA and the capsular region of CEA (Fig. 5D, E). The pattern of labeling caudally in the diencephalon (Fig. 6Q–S) was similar to that seen rostrally; that is,

essentially confined to PRC and to the amygdala. Labeled fibers spread to all layers of PRC but were most densely concentrated in inner layers. A few fibers extended ventrally from PRC to the entorhinal cortex. At the caudal amygdala, BLA was moderately labeled and the postpiriform area lightly labeled (Fig. 6R, S).

DISCUSSION Using an anterograde tracing technique, we examined the efferent projections of the central medial nucleus of the intralaminar thalamus in the rat. CM predominantly targets the prefrontal/frontal cortex, the dorsal and ventral striatum and the amygdala, distributing heavily to these sites (Fig. 8). Rostral and caudal CM project commonly to several structures, but to different regions of these structures. In effect, there is relatively little overlap in the two sets of projections (CMr and CMc) (Fig. 8). CM output to limbic and sensorimotor structures of the rostral forebrain

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Fig. 6. Schematic depiction of labeling present in representative sections through the forebrain (A–S) produced by a PHA-L injection (red dots in Q, R) in the caudal part of the central medial nucleus of thalamus (case 32). Sections modified from the rat atlas of Swanson (2004). See list for abbreviations. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6 (continued)

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Fig. 6 (continued)

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Fig. 7. (A–C) Low-power bright-field photomicrographs of three rostrocaudally aligned transverse sections through the rostral forebrain showing patterns of labeling produced by a PHA-L injection in the caudal part of the central medial nucleus. Note the dense labeling ventrolaterally within the anterior cortex, localized to the DLO, rostrally (A) and to the dorsal agranular insular cortex, caudally (B, C), mainly concentrated in layers 1 and 3 of these cortical regions. Note also pronounced labeling in the prelimbic cortex (A) and the anterior claustrum (C). (D, E) Low-power bright-field photomicrographs of a rostral (D) and caudal (E) transverse sections through the forebrain showing patterns of labeling in the dorsal striatum produced by a PHA-L injection in the caudal part of the central medial nucleus. Note dense aggregates of labeled fibers ventrally/ventrolaterally in CP, rostrally (D) and medially along the lateral border of the globus pallidus, caudally (E). Note also pronounced labeling in the posterior agranular insular and visceral cortices (E). Scale bar for A, D = 500 lm; for B = 310 lm; for C = 750 lm; for E = 400 lm. See list for abbreviations.

suggests that CM may serve to integrate affective, cognitive and sensorimotor functions for goal-directed behavior. A note on potential uptake by fibers of passage With all anterograde tracers, there is the possibility of uptake of tracers not only by cells at the site of injection but also by fibers passing through the injection – the fibers of passage problem. This is particularly relevant here since the intralaminar nuclei are embedded in the fibers of the internal medullary lamina. Of the anterograde tracers, however, PHA-L appears to be the least susceptible to uptake by coursing fibers (Gerfen and Sawchenko, 1984; Groenewegen and Wouterlood, 1990). Specifically, PHA-L requires ionotophoretic application and is only successfully transported by cells that have incorporated the

tracer, that is, not by cells/fibers in the zone of passive spread of the tracer (Gerfen and Sawchenko, 1984). In this regard, we saw no evidence of transport from regions adjacent to CM which were sometimes included in the area of passive spread of the tracer such as the intermediodorsal (IMD), paracentral or rhomboid nuclei. On rare occasions we have observed the retrograde transport of PHA-L (suggesting some uptake by passing fibers), but this generally only occurs with large PHA-L injections and was not presently seen. In addition, previous studies using PHA-L have described distinct and largely nonoverlapping midline/ILt thalamic projections to the striatum and cortex (Berendse and Groenewegen, 1990, 1991), despite the fact that injection sites for some cell groups are traversed by fibers from other ILt nuclei en route to their targets (Berendse and Groenewegen,

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Cortex

CMr

medial AC AGm PL IL RSC lateral AGl LO VLO DLO AId AIp GU SSI SSII PRC ECT EC

Fiber Density light moderate heavy

CMc

Basal Forebrain ACC CLA C-P OT

Amygdala AAA BLA BMA LA CEA COAa

Fig. 8. Summary diagram of the pattern of projections of the rostral (CMr) and the caudal (CMc) central medial nucleus of the thalamus to its main targets; namely, the cerebral cortex (medial and lateral regions), the basal forebrain and the amygdala. The relative density of projections to each structure is indicated by the thickness of the lines. See list for abbreviations.

1990). Accordingly, the uptake of PHA-L by fibers coursing through CM was likely minimal. Overview of CM projections CMr projections. The main CMr targets were rostral regions of the cortex, the CLA, dorsal striatum, ACC, OT, and BLA. Some secondary sites were the posterior insular and perirhinal cortices. With respect to the cortex, CMr distributes to rostral but essentially not to caudal regions of the cortex, densely innervating the AGm, anterior cingulate, prelimbic, DLO and AId cortices. CMr projects much more heavily to the dorsal (AGm and AC) than to the ventral mPFC (PL and IL). CMr distributes massively throughout rostrocaudal extent of the dorsal striatum, mainly targeting central regions of C-P, rostrally, and the dorsal/dorsomedial half of C-P, caudally. Within the ventral striatum, CMr fibers were virtually confined to the anterior ACC, terminating substantially in the rostral pole, core and shell regions of the rostral ACC. Finally, CMr fibers distribute throughout the basolateral nucleus of the amygdala, virtually blanketing BLA. With the exception of lateral half of the rostral pole of BLA, the entire nucleus was densely labeled. CMc projections. Major CMc termination sites were lateral regions of the anterior cortex, the CLA, the dorsal

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striatum, the posterior agranular insular cortex, the PRC and the amygdala. CMc fibers generally avoided the mPFC, distributing substantially instead to lateral parts of the prefrontal cortex including the ventrolateral, lateral and dorsolateral orbital cortices, the dorsal (AId) and ventral agranular insular cortices, gustatory/VISC cortex, and the primary somatosensory (SSI) and motor (AGl) cortices. Of these regions, AId, gustatory and SSI cortices were densely labeled; AGl was slightly less heavily labeled. At caudal levels of the cortex, CMc fibers were mainly localized to regions bordering (and dorsal to) the rhinal fissure including the posterior agranular insular, PRC, and visceral cortices. CMc distributes heavily to the dorsal striatum, with fibers predominately targeting the ventral/ventrolateral quadrant of C-P, rostrally, and the dorsoventral expanse of the medial C-P, caudally. CMc projects sparsely to the rostral C-P. CMc fibers distribute widely throughout the amygdala reaching most subnuclei. These include regions of the anterior, lateral (LA), central (CEA), medial, cortical and basal (BLA and BMA) nuclei of amygdala, with most pronounced projections to LA, capsular CEA, anterior cortical, and BMA. Major CM projections: comparisons with previous studies Projections to the cortex. As described, CMr and CMc distribute to essentially non-overlapping regions of the cortex. In a review, Van der Werf et al. (2002) described similar findings, stating: ‘‘There is a striking difference in the pattern of terminal labeling following rostral or caudal injections in the CeM. At cortical levels, virtually no overlap can be seen between the termination fields resulting from either rostral or caudal injections.’’ CMr projections to cortex. We showed that CMr projections to the cortex were virtually restricted to anterior regions of the cortex. In an early examination of midline/ ILt thalamic projections to cortex in rats, Berendse and Groenewegen (1991) similarly reported that the rostral CM targets the anterior PFC, but unlike present findings, described more limited CMr projections to the cortex, or mainly confined to AC and to anterior PL. Few fibers were identified in AGm and virtually none in DLO or AId. Differences may involve differing locations of injections in CMr. Supporting present findings of a pronounced CMr input to AGm, Reep and Corwin (1999) described substantial numbers of labeled cells in rostral CMr following retrograde injections in the rostral AGm of rats. In like manner, we previously showed that retrograde injections in the dorsal mPFC (AGm and AC) of rats produced robust cell labeling in CM, predominantly localized to CMr, whereas those in the ventral mPFC (PL and IL) gave rise to few labeled neurons in CM (Hoover and Vertes, 2007). CMc projections to cortex. As described, CMc distributes heavily to lateral regions of the cortex – around and dorsal to the rhinal fissure. An early report by Reep and Winans in rats (1982) described labeled cells in CM (mainly in CMc) with retrograde injections in AId, but

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few with injections in AIv. Jasmin et al. (2004) subsequently demonstrated pronounced cell labeling in CM, mainly restricted to CMc, following injections in the rostral agranular insular cortex (AId + AIv) of rats. Finally, Guldin and Markowitsch (1983) reported that injections of retrograde tracers in the PRC give rise to labeled cells confined to the midline/ILt thalamus, mainly to CMc. Projections to the dorsal striatum (caudate-putamen) We showed that CMr and CMc distribute massively to the dorsal striatum and to largely separate regions of C-P. An early examination of thalamic afferents to the striatum (Berendse and Groenewegen, 1990) described a similar pattern of CMr projections to C-P in rats, but less widely distributed throughout the striatum than shown here – or as we demonstrated (Fig. 2) to most of the medial/central C-P, rostrally, and throughout the dorsal half of C-P, caudally. In partial support of the present findings, Van der Werf et al. (2002) demonstrated that CMc fibers target the ventrolateral quadrant C-P – or the fundus of the striatum. Unlike their findings, however, we showed that CMc fibers are not confined to the fundus but spread throughout the ventrolateral C-P, densely concentrated along the lateral border of GP. Finally, retrograde tracer injections in C-P gave rise to labeled cells in several midline/ILt nuclei in rats including CM, with the strongest CM labeling with injections in the dorso-central and ventrolateral C-P (Erro et al., 2002). Projections to the ventral striatum (nucleus accumbens) We showed that: (1) CMr but not CMc projects to ACC; and (2) CMr distributes heavily to the rostral pole and core of ACC, and moderately to lateral parts of the medial shell of ACC. Several previous reports in rats have shown that the midline/ILt thalamus is a major source of projections to ACC, mainly originating from the paraventricular (PV), paratenial (PT), IMD, and CM nuclei (Groenewegen et al., 1980; Phillipson and Griffiths, 1985; Berendse et al., 1988; Berendse and Groenewegen, 1990; Su and Bentivoglio, 1990; Brog et al., 1993; Van der Werf et al., 2002; Li and Kirouac, 2008; Vertes and Hoover, 2008). Consistent with present findings, Berendse and Groenewegen (1990) demonstrated that retrograde injections in the core but not the shell of ACC gave rise to robust labeling in CM and that anterograde injections in CMr produced strong terminal labeling in the rostral core of ACC. In accord with this, Brog et al. (1993) described a progressive decrease in numbers of labeled cells in CM with retrograde injections in the core, lateral shell and medial shell of ACC.

afferents to the amygdala from several nuclei of thalamus, mainly those of the midline/ILt thalamus. Of the latter, projections were heaviest from the PV, PT, interanteromedial and parafascicular nuclei to the amygdala, mainly to BLA. CM projections to the amygdala were modest. By contrast, Su and Bentivoglio (1990) demonstrated moderately retrograde cell labeling in CM following large amygdaloid injections centered in BLA in rats; while Turner and Herkenham (1991) described dense CM projections to the amygdala selectively targeting BLA – and heavier to the anterior than to the posterior BLA.

Central medial nucleus: an integral component of a midline thalamic network with widespread influence on the limbic forebrain Although CM is part of the rostral ILt (Van der Werf et al., 2002), it lies on the midline and as such appears to share characteristics with the midline thalamic nuclei. As a group, the midline thalamic nuclei (and CM) receive a diverse and widespread set of afferent projections and predominately target limbic forebrain structures (Van der Werf et al., 2002; Vertes, 2006). Accordingly, the midline nuclei appear to represent an important interface between ‘lower’ and ‘higher’ levels of the limbic system in affective and cognitive control (Vertes, 2006). Although partially overlapping, each of the midline thalamic nuclei projects to unique sets of limbic forebrain structures. For instance, the reuniens and rhomboid nuclei of the ventral midline thalamus primarily target limbic cortical structures, prominently including the hippocampus and mPFC (Herkenham 1978; Wouterlood et al., 1990; Wouterlood, 1991; Dollerman-Van der Weel and Witter, 1996; Risold et al., 1997; Bokor et al., 2002; Vertes, 2006; Vertes et al., 2006), whereas the paraventricular and paratenial nuclei of the dorsal midline thalamus mainly distribute to limbic subcortical sites in rats and primates (Li and Kirouac, 2008; Vertes and Hoover, 2008; Hsu and Price, 2009). By comparison, the central medial nucleus (lying between the dorsal and ventral midline groups) projects more uniformly to subcortical and cortical limbic sites. In this respect, CM may bridge the dorsal and ventral midline thalamus, simultaneously influencing subcortical and cortical structures. Finally, unlike other midline nuclei, CM is a major source of afferents to the dorsal striatum suggesting that CM integrates sensorimotor and limbic functions. In summary, CM appears ideally positioned to integrate the activity of its main targets, the prefrontal cortex, the dorsal and ventral striatum and the amygdala, for goal-directed behavior. In effect, CM may promote the temporary storage of information (working memory) at the PFC, its evaluation at the amygdala and nucleus accumbens, and preparation for actions at the striatum.

Projections to the amygdala We showed that CMr virtually exclusively targets BLA of amygdala, whereas CMc fibers distribute throughout the amygdala terminating in the lateral, central, anterior cortical and basomedial nuclei of amygdala. In an early examination of thalamo-amygdaloid projections in the rat, Ottersen and Ben-Ari (1979) described

Acknowledgements—The present study was supported by NSF grant IOS 0820639 to RPV and by the Government of the Basque Country grant (AE-2010-1-28; AEGV10/16) and the grant agency of the Czech Republic (GACR 309/09/1696) to JJR. The authors sincerely thank two anonymous reviewers for their very constructive comments on earlier versions of the manuscript.

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(Accepted 29 April 2012) (Available online 7 May 2012)