Bed nucleus of the stria terminalis and extended amygdala inputs to dopamine subpopulations in primates

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Neuroscience Vol. 104, No. 3, pp. 807^827, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522 / 01 $20.00+0.00

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BED NUCLEUS OF THE STRIA TERMINALIS AND EXTENDED AMYGDALA INPUTS TO DOPAMINE SUBPOPULATIONS IN PRIMATES J. L. FUDGEa;b and S. N. HABERb;c * a

Department of Psychiatry, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, NY 14642, USA

b

Department of Neurobiology and Anatomy, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, NY 14642, USA

c

Department of Neurology, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, NY 14642, USA

AbstractöThe `extended amygdala', a forebrain continuum implicated in complex motivational responses, is comprised of the bed nucleus of the stria terminalis and its sublenticular extension into the centromedial amygdala. Dopamine is also involved in motivated behavior, and is increased in several brain regions by emotionally relevant stimuli. To examine how the extended amygdala in£uences the dopamine cells, we determined the organization of inputs from subdivisions of the bed nucleus of the stria terminalis and sublenticular extended amygdala to the dopamine subpopulations in monkeys. Inputs from the bed nucleus of the stria terminalis and corresponding regions of the sublenticular extended amygdala are di¡erentially organized. The medial bed nucleus of the stria terminalis and its medial sublenticular extension have a mediolateral organization with the densest inputs to the medial substantia nigra, pars compacta, and relatively few inputs to the central and lateral substantia nigra. In contrast, the lateral bed nucleus of the stria terminalis (and its continuation into the sublenticular extended amygdala) projects across the mediolateral extent of the substantia nigra. The subnuclei of the lateral bed nucleus of the stria terminalis also have di¡erential projections to the dopamine cells. While the central core of the lateral bed nucleus of the stria terminalis has restricted inputs, the surrounding dorsolateral, capsular and juxtacapsular subdivisions project strongly to the dorsal tier dopamine neurons. The posterior subdivision of the lateral bed nucleus of the stria terminalis and its continuation into the central sublenticular extended amygdala project more broadly to both the dorsal tier and densocellular region of the ventral tier. From these results we suggest that speci¢c subdivisions of the bed nucleus of the stria terminalis have di¡erential in£uences on the dopamine subpopulations, in£uencing dopamine responses in diverse brain regions. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: extended amygdala, dopamine, substantia nigra, ventral tegmental area, primate, bed nucleus of the stria terminalis.

emotionally relevant stimuli. Behavioral studies show that both the BST and the CeN are important in orienting to unpredicted stimuli and in endocrine and behavioral responses to stress (Henke, 1984; Gray, 1993; Holland and Gallagher, 1993; Menzaghi et al., 1993; Davis et al., 1994; Gallagher and Chiba, 1996; Lee and Davis, 1997). The extended amygdala is linked to the midbrain dopamine (DA) cells by a direct a¡erent connection. Consistent with this, behavioral studies indicate that novel and stressful stimuli cause DA increases in speci¢c brain regions (Thierry et al., 1976; Abercrombie et al., 1989; Abercrombie and Zigmond, 1995; Kalivas and Du¡y, 1995; Feenstra and Botterblom, 1996; Rebec et al., 1997a; Inglis and Moghaddam, 1999). Thus, one important way that unpredicted and stress-related stimuli can directly in£uence DA cell activity is through input from the extended amygdala. The DA neurons are organized into the dorsal and ventral tiers based on morphological, histochemical, and connectional criteria (Lavoie and Parent, 1991; Hirsch et al., 1992; Lynd-Balta and Haber, 1994b; Haber et al., 1995; McRitchie and Halliday, 1995). The `dorsal tier' includes the calbindin-Dk28, (CaBP)-positive ventral tegmental area (VTA), contiguous dorsal substantia nigra, pars compacta (SNc), and lateral dorsal

The extended amygdala, which includes the bed nucleus of the stria terminalis (BST) and central amygdaloid nucleus (CeN), is hypothesized to mediate responses to

*Corresponding author. Tel.: +1-716-275-6959; fax: +1-716-7565334. E-mail address: [email protected] (S. N. Haber). Abbreviations : ABC, avidin^biotin^peroxidase complex ; AChE, acetylcholinesterase ; BST (L, LD, LC, Lcn, LJ, LP, LS, M, MS), bed nucleus of the stria terminalis (lateral, lateral dorsal, lateral capsular, lateral central, lateral juxtacapsular, lateral posterior, lateral supracapsular, medial, medial supracapsular); CaBP, calbindin-Dk28, Ca2‡ binding protein ; CeLc, central amygdaloid nucleus, capsular subdivision; CeLcn, central amygdaloid nucleus, lateral subdivision ; CeN, central amygdaloid nucleus; DA, dopamine; DAB, 3,3P-diaminobenzidine ; HRPWGA, horseradish peroxidase conjugated to wheat germ agglutinin ; LY, Lucifer Yellow conjugated to dextran amine; NBM, nucleus basalis of Meynert; NGS, normal goat serum; PB, phosphate bu¡er; PB-T, phosphate bu¡er with Triton X-100 ; PHAL, phaseolus vulgaris leucoagglutinin; SLEA(c,m), sublenticular extended amygdala (central, medial division); SN(c,r), substantia nigra (pars compacta, reticulata); TH, tyrosine hydroxylase; VTA, ventral tegmental area. 807

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SNc (including the retrorubal ¢eld). The dorsal tier neurons receive `limbic-related' inputs (Lynd-Balta and Haber, 1994c; Fudge and Haber, 2000; Haber et al., 2000) and project to the ventral striatum, amygdala, and cortex (Fallon, 1978; Fallon and Moore, 1978; Porrino and Goldman-Rakic, 1982; Deutch et al., 1988; Gaspar et al., 1992; Lynd-Balta and Haber, 1994b), where DA levels increase in response to reward, stress, and novelty (Kalivas and Du¡y, 1995; Feenstra and Botterblom, 1996; Richardson and Gratton, 1996; Rebec et al., 1997b; Richardson and Gratton, 1998; Harmer and Phillips, 1999; Inglis and Moghaddam, 1999). In contrast, the CaBP-negative `ventral tier' neurons receive inputs from limbic, cognitive, and sensorimotor striatal domains, and project back to all striatal territories through a series of feed-forward loops (Haber et al., 2000). The extended amygdala is divided into `central' and `medial' components. The central extended amygdala includes the CeN, the central sublenticular extended amygdala (SLEAc), and the lateral division of the BST (BSTL). The BSTL and CeN form the rostral and caudal poles, respectively, of the central extended amygdala, and have a number of similarities (DeOlmos et al., 1985; Alheid et al., 1995). Like the CeN, the BSTL is a complex region with several subdivisions, which bear striking structural and chemical similarities to those in the central nucleus (McDonald, 1982; McDonald, 1983; DeOlmos et al., 1985; Cassell and Gray, 1989; Moga et al., 1989; Shimada et al., 1989; DeOlmos, 1990; Martin et al., 1991; Sun and Cassell, 1993; Heimer et al., 1999). The medial extended amygdala includes the medial amygdaloid nucleus and its cellular extension to the medial BST (BSTM). While previous studies have shown that the BST and CeN both project to the DA cells (Conrad and Pfa¡, 1976; Meibach and Siegel, 1977; Krettek and Price, 1978; Phillipson, 1979; Swanson, 1979; Price and Amaral, 1981; Morrell et al., 1984; Holstege et al., 1985; Grove, 1988a; Gonzales and Chesselet, 1990; Vankova et al., 1992; Wallace et al., 1992), there is little information on how speci¢c subdivisions of the extended amygdala in£uence the DA cells. In examining the projections of the CeN which forms the caudal end of this continuum, we have recently found that speci¢c CeN subdivisions project di¡erentially to subpopulations of DA neurons. This organization provides an anatomic substrate for the di¡erential regulation of speci¢c DA output pathways (Fudge and Haber, 2000). This study extends these ¢ndings to explore the organization of inputs to the DA cells from the other components of the extended amygdala, namely the BST and the SLEA.

EXPERIMENTAL PROCEDURES

Injection sites Ten injections of horseradish peroxidase conjugated to wheat germ agglutinin (HRP-WGA) or Lucifer Yellow conjugated to dextran amine (LY) were made to speci¢c regions of the ventral midbrain. Injections were placed medially (encompassing both

the SN and VTA), centrally, and laterally. Three injections were placed caudal to the rami¢cation of the third nerve fascicles, in the central and lateral SN. Each injection site was analyzed with respect to its position within the dorsal and ventral tiers as described previously (Fudge and Haber, 2000). To con¢rm retrograde data, the anterograde tracers phaseolus vulgaris leucoagglutinin (PHAL) and LY were placed in the BSTL and dorsal SLEA, respectively. Anterogradely labeled ¢bers in the midbrain were analyzed with respect to their distribution within the DA subpopulations. Combinations of tracers known to have cross-reactive antibodies were not used in the same animal. Experimental procedures Twelve Old World monkeys (Macaca nemestrina), purchased from the University of Washington Regional Primate Research Center (Seattle, WA, USA), were used in these experiments. All experiments were carried out in accordance with National Institutes of Health guidelines. Experimental design and techniques aimed at minimizing animal use, and animal su¡ering were reviewed and approved by the University of Rochester Committee on Animal Research. Initial anesthesia via an intramuscular injection of ketamine (10 mg/kg) was followed by a deep surgical level of anesthesia using pentobarbital (initial dose 20 mg/kg with maintenance dosing as needed). The injection sites were located using electrophysiologic mapping (Haber et al., 1985). HRP-WGA (10%, Sigma, St. Louis, MO, USA) and LY (10%, Molecular Probes) were pressure-injected (35^40 nl) into discrete regions of the ventral midbrain using a Hamilton 0.5 Wl syringe (Hamilton, Reno, NV, USA). Two injections were made using glass pipet tips (approximately 50 Wm diameter) placed over the syringe tip. The anterograde tracers PHAL (2.5%, Vector Laboratories, Burlingame, CA, USA) and LY (10%, Molecular Probes) were pressure-injected into the lateral extended amygdala trajectory at di¡erent levels. Seven to 10 days after surgery the animals were again deeply anesthetized and killed by intracardiac perfusion using saline followed by a 4% paraformaldehyde solution in 0.1 M phosphate bu¡er (PB), pH 7.4. The brains were cryoprotected in increasing gradients of sucrose (10, 20, and 30%). Serial sections of 50 Wm were cut on a freezing microtome and processed for immunocytochemistry for HRP-WGA, LY, or PHAL. Sections were ¢rst rinsed in 0.1 M PB (pH 7.4) with 0.3% Triton X-100 (PB-T) and then preincubated in 10% normal goat serum (NGS) diluted with PBS-T (NGS-PB-T) for 30 min. Tissue was then placed in the primary antisera anti-HRP-WGA 1:2000 (Sigma, St. Louis, MO, USA) or anti-LY 1:2000 (Molecular Probes) in NGS-PBS-T for four nights (approximately 96 h) at 4³C. Tissue processed for PHAL was placed in antisera (1:500, Molecular Probes) for six nights (approximately 144 h). The avidin^biotin^peroxidase complex (ABC) reaction (rabbit Vectastain ABC kit, Vector) was used to visualize all three tracers. Tissue was incubated for 10^12 min in 3,3P-diaminobenzidine tetrahydrochloride (DAB) and 0.03% H2 O2 , and then intensi¢ed with 1% cobalt chloride and 1% nickel ammonium sulfate to yield a black reaction product. Retrograde cases. CaBP was used as a marker of the dorsal tier. In order to determine the relative placement of retrograde injection sites within the dorsal and ventral tiers, midbrain sections were double labeled for the tracer molecule and for CaBP immunoreactivity. Sections were ¢rst processed for tracer and intensi¢ed to a black reaction product. The tissue then underwent immunohistochemical staining for CaBP. Sections were ¢rst rinsed in 0.1 M PB-T and then preincubated in 10% NGS-PB-T for 30 min. Tissue was then placed in the primary antisera anti-CaBP 1:10 000 (mouse monoclonal, Sigma) in NGS-PB-T for four nights (approximately 96 h) at 4³C. The ABC reaction was used to visualize CaBP-positive regions. Tissue was incubated for 10^12 min in DAB and 0.03% H2 O2 to yield a light brown reaction product. To determine the subdivisions of the BST and to distinguish this region from the adjacent striatum, we double-labeled sections for the tracer molecule and acetylcholinesterase (AChE)

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staining (Gaspar et al., 1985; DeOlmos, 1990) using a modi¢ed Geneser technique (Geneser-Jensen and Blackstad, 1971) (see Results). The distribution of retrogradely labeled cells within the BST subdivisions was then charted. In order to determine the distribution of labeled cells in the SLEAc, some compartments were double-labeled for the tracer molecule and CaBP. CaBP-positive ¢bers are found in the pallidum, but not in the adjacent SLEA (Cote et al., 1991). In addition, the boundary between the SLEA and globus pallidus was demarcated by the distribution of iron deposits concentrated over the pallidum, seen in DAB-reacted sections. To delineate the distribution of retrogradely labeled cells in medial and central extended amygdala, adjacent compartments were also processed for enkephalin immunoreactivity (1:1000, the generous gift of Dr. Robert Elde, University of Minnesota, MN, USA) which is high in the central extended amygdala and relatively low in medial extended amygdala (Lesur et al., 1989; Martin et al., 1991; Heimer et al., 1999). Anterograde cases. The location of the anterograde injection site in case 39 was determined using an AChE counterstain. The placement of the injection site in case 42 was determined using double-immunohistochemistry for tracer and met-enkephalin. In both cases, additional compartments were processed for the tracer molecule and counterstained with Cresyl Violet. In order to identify the DA cells in midbrain sections labeled for anterograde tracer, the tissue was secondarily immunoreacted for tyrosine hydroxylase (TH). After processing sections for tracer using the nickel intensi¢cation technique (described above), they were incubated in anti-TH 1:10 000 (Eugene Tech, Eugene, OR, USA) for four nights, then processed using the ABC reaction (Vectastain Elite kit, Vector) to yield a light brown reaction product in TH-positive regions. Adjacent compartments were processed for CaBP immunohistochemistry to determine the boundaries of the dorsal and ventral tiers. Analysis We eliminated from the analysis all injection sites in which there was necrosis at the injection site or leakage of tracer into surrounding structures. Charts were drawn with a Leitz diaplan microscope (25U objective) with a drawing tube attachment, and entered into the computer using the drawing program Canvas 5.0. Nuclear boundaries and subdivisions were also charted and entered into the computer. To assess for ¢bers of passage problem, we examined additional cases in which retrograde tracer injections missed the SNc target, and were situated in surrounding ¢ber tracts. For anterograde cases, labeled ¢bers and landmarks in each midbrain section were charted using bright and dark-¢eld microscopy. Charted sections were then scanned into the computer on a UMAX PowerlookIII scanner at 600 dpi, converted to a bit-map, and transferred into the graphics program Adobe Illustrator. CaBP-positive cells were charted and superimposed onto charted sections of labeled ¢bers and TH-positive cells. Using these images, the distribution of labeled ¢bers in the dorsal (CaBP-positive) and ventral (CaBP-negative) tiers was analyzed.

809 RESULTS

De¢ning the regions Subdivisions of the BST (Fig. 1). The primate BST subdivisions, and in particular the components of the BSTL, have been described using various terminologies (Gaspar et al., 1985; Lesur et al., 1989; DeOlmos, 1990; Martin et al., 1991; Walter et al., 1991; Kaufmann et al., 1997; Heimer et al., 1999). We refer to the BST subdivisions (as well as the CeN subdivisions) using the nomenclature proposed by Heimer et al. (1999). In the primate, the BSTL is relatively more developed and di¡erentiated compared to the BSTM. The BSTL has heterogeneous AChE staining compared to the BSTM, which contains relatively low AChE levels (Gaspar et al., 1985). Rostrally, the region directly adjacent to the striatum is the lateral dorsal BST (BSTLD). The BSTLD encircles the emerging anterior commissure medially and ventrally. At its dorsal boundary with the ventromedial striatum, the BSTLD is distinguished by its lower AChE levels (Fig. 1A,AP). The lateral juxtacapsular subdivision of the BST (BSTLJ) forms a strip of relatively high but heterogeneous AChE staining laterally (Fig. 1A,AP,B,BP). Further caudal, the BSTLJ forms a longitudinal stripe medial to the internal capsule (Fig. 1B,BP). The BSTLJ has been linked to the intercalated cell islands of the amygdala because it also contains clusters of granular cells (McDonald, 1983; Martin et al., 1991). At this level, the BST lateral central division (BSTLcn) emerges and is evident by its low^moderate AChE staining, and its loosely packed, homogeneous cells as seen in Nissl-stained sections. The BSTLcn is surrounded by the ¢brous lateral central BST (BSTLC), which is relatively devoid of AChE staining. Enkephalin staining at this level reveals that the BSTLcn contains many enkephalin-positive cells and a dense meshwork of ¢ne, punctate enkephalin-positive ¢bers (Fig. 1C,D,F). The BSTLC is invaded by enkephalinpositive `woolly-type' ¢bers similar to those seen in the globus pallidus (Haber and Watson, 1985) (Fig. 1E). Lateral posterior BST (BSTLP) emerges caudal to the other lateral subdivisions and sweeps medially and ventrally around the BSTLcn (Fig. 1B,C). It also contains `woolly-type' ¢bers. The BSTLP has relatively low AChE levels compared to the other lateral subdivisions and merges with the SLEAc. The supracapsular BST is con-

Abbreviations used in the ¢gures AC Astr C CL DT F GP(e, i) HBD IC IP

anterior commissure amygdalostriatal area caudate nucleus claustrum dorsal tier fornix globus pallidus (external, internal segment) horizontal limb of the diagonal band of Broca internal capsule interpenduncular nucleus

M OT PL RRF S ST Th V VP VT

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medial nucleus optic tract substantia nigra, pars lateralis retrorubal ¢eld septum stria terminalis thalamus ventricle ventral pallidum ventral tier

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Fig. 1.

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Fig. 2. (A, B) Enkephalin staining in the SLEAc in coronal sections at two rostrocaudal levels. Arrows indicate enkephalinergic ¢bers and terminals in the SLEAc. The supracapsular BST (A) contains relatively higher enkephalin staining in the BSTLS compared to the BSTMS.

sidered the caudal extent of the BST, and is located beneath the body of the caudate nucleus. Lateral supracapsular BST (BSTLS) is found dorsolaterally, and contains relatively higher enkephalin staining than the medial supracapsular subdivision (BSTMS) (see Fig. 2A). SLEA (Fig. 2). The SLEA is divided into two regions: medial and central. The medial SLEA (SLEAm) forms a cellular bridge between the BSTM rostrally, and the medial amygdala nucleus caudally. Likewise, the SLEAc bridges the gap between the BSTL rostrally and the central amygdala nucleus caudally. The SLEAc contains intermediate levels enkephalin-positive immunoreactivity (Fig. 2A,B, arrowheads) and AChE staining. Adjacent to the anterior SLEAc lies the ventral pallidum, which also contains many enkephalin-positive ¢bers. However, in contrast to the SLEAc, the ventral pallidum is demarcated by CaBPpositive ¢bers, not found in the SLEAc (see Fig. 1F). Further caudal (Fig. 2B), the posterior SLEAc contains light to moderate enkephalin staining. Enkephalin-positive `woolly' ¢bers invade the SLEAc beneath the pallidum and continue into the central nucleus, mainly in the medial subdivision (see Haber and Watson, 1985). In contrast to the SLEAc, SLEAm contains relatively weak levels of enkephalin immunoreactivity and AChE staining. DA subpopulations (Fig. 3A^D). The SNc is divided into the dorsal and ventral tiers. The continuity of the dorsal tier at all rostrocaudal levels is characterized by 6

several features including horizontally oriented dendrites and CaBP immunoreactivity (Fallon and Moore, 1978; Poirier et al., 1983; Francois et al., 1987; Yelnik et al., 1987; Lavoie and Parent, 1991; McRitchie and Halliday, 1995; Francois et al., 1999). Furthermore, the entire rostrocaudal and mediolateral extent of the dorsal tier receives a¡erents from `limbic-related' structures such as the shell of the ventral striatum and the CeN (LyndBalta and Haber, 1994c; Fudge and Haber, 2000; Haber et al., 2000). Based on these criteria, the dorsal tier includes the rostrocentral VTA and contiguous dorsal SN (Fig. 3A,B), and the caudal VTA, retrorubal ¢eld, and lateral dorsal SNc (Fig. 3C,D). The ventral tier, comprised of the main densocellular region and vertically oriented cell columns, is CaBP-negative (arrows). The ventral tier receives broad striatal a¡erents which terminate across its rostrocaudal extent, forming an inverse dorsoventral gradient (Szabo, 1970; Hedreen and DeLong, 1991; Lynd-Balta and Haber, 1994c; Haber et al., 2000). The ventrolateral SN, also known as the `pars lateralis' (Francois et al., 1984; Yelnik et al., 1987; Juraska et al., 1977), contains columns of TH-positive cell bodies, and few to no CaBP-positive cells. We therefore consider this region as part of the ventral tier. Location of injection sites Ten injection sites were located in the midbrain. Five injection sites included the dorsal tier, and ¢ve had little to no inclusion of the dorsal tier (Fig. 4A,B). Of the ¢ve dorsal tier injections, one (case 25) was con¢ned to the

Fig. 1. (A^C) Schematics of the coronal sections through the BST and SLEA at three rostrocaudal levels. (AP,BP) Photomicrographs of AChE-stained sections corresponding to A and B. (D) Coronal section through the BST and anterior SLEAc stained for met-enkephalin. (E) High power photomicrograph showing ¢ne, punctate enkephalinergic ¢bers and `woolly-type' enkephalinergic ¢bers (ENK arrows) in the BST lateral capsular (BSTLC) and lateral posterior (BSTLP) (small boxed area in D). Several enkephalin-positive cell bodies are also seen (white arrowheads). (F) The boundary between the ventral pallidum (CaBP-positive) and the SLEAc (CaBP-negative) in a section adjacent to D (large boxed area in D).

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Fig. 3. Photomicrographs of adjacent coronal sections through the ventral midbrain at two rostrocaudal levels. (A, C) TH immunostaining ; (B, D) CaBP immunostaining. The CaBP-positive cellular regions delineate the dorsal tier, and include the retrorubal ¢eld. Arrowheads indicate CaBP-negative areas of the ventral tier.

dorsal tier in the region of the VTA, three included relatively more of the dorsal tier but extended into the densocellular area (cases 67, 72R, and 72L), and one large medial injection (case 68) included the dorsal tier but was centered in the densocellular region. Of the ¢ve injection sites centered in the ventral tier, two were centered on the densocellular region (cases 48, 72LY) and extended into the cell columns. Three injection sites were centered on the cell columns/pars reticulata with little to no encroachment on the densocellular region (cases 62, 68L, 69). There were two anterograde tracer injection sites located in the central extended amygdala. Both anterograde injection sites were characterized by a relative absence labeled ¢bers in the subthalamic nucleus, or in the internal and external segments of the globus pallidus, which receive pallidal a¡erents (Zahm, 1989; Haber et al., 1993). One injection site was located in the BSTLP (case 39), and one injection site was located in the posterior SLEAc (case 42) (Fig. 4C). Injection sites centered in the dorsal tier Case 25: Labeled cells were found in both divisions of the BST and SLEA, £owing in a continuous stream to the centromedial amygdala. The majority of labeled cells

were in the BSTL, however, a moderate number of labeled cells were also seen in the BSTM. Two main streams of labeled cells could be seen through the forebrain, consistent with the general organization of the SLEA into central and medial divisions. At rostral levels (Fig. 5A), the BSTLD contained clusters of labeled cells which were particularly dense dorsomedially and extended into the striatum (Fig. 5D, DP). Labeled cells were also prominent beneath the commissure and continued in ¢bers along the ventral border of the internal capsule and under the pallidum. There were also many HRP-positive cells in the BSTM at this level. Further caudal (Fig. 5B, E), there was a high concentration of labeled cells in the BSTLJ, which appeared to merge with the BSTLC. HRP-positive cells in the BSTLC encircled the BSTLcn, which contained scattered labeled cells dorsally. Many labeled cells were seen in the BSTLP ventrally, and continued underneath the pallidum in the SLEAc. Labeled cells in the BSTM were concentrated lateral to the fornix and continued into the SLEAm (Fig. 5B) where they merged with cells in the anterior hypothalamus (not shown). At caudal levels, there were scattered HRP-positive cells in the BSTLS, and none in the BSTMS (Fig. 5C). Case 68R: Labeled cells were seen in both the BSTM/

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Fig. 4. Placement of injection sites. (A) Retrograde injection sites in the ventral midbrain at rostrocentral and caudal levels. Sites in dark gray include the dorsal tier; sites in medium gray are centered on the densocellular region with little encroachment on the dorsal tier, and sites in light gray are centered on the cell columns and pars reticulata. (B) Photomicrographs of injection sites for cases 48, 69, 72LY. (C) Photomicrographs of injection sites for anterograde cases 39 (PHAL) and 42 (LY). Note : the staining of several electrode tracks results from DAB reaction to local injury, and is not due to tracer leakage.

SLEAm and BSTL/SLEAc, but were more moderately concentrated than in case 25 (above) despite the relatively large size of the injection site. Rostrally, labeled cells were moderately concentrated in the BSTLD (Fig. 6A). Labeled cells in the BSTM continued into the lateral septum (not shown). At somewhat more caudal levels, labeled cells were mainly seen in both the BSTLP and SLEAc (Fig. 6B). The BSTLC and BSTLJ contained a light distribution of labeled cells, and there were few to no labeled cells in the BSTLcn. Further caudal (Fig. 6C), labeled cells in the SLEAc coursed among the large AChE-positive cells of the nucleus basalis of Meynert (NBM) most of which were not themselves labeled for tracer. A ventromedial stream of labeled cells emerged beneath the internal capsule and continued ventrally along the base of the forebrain in the SLEAm. There were scattered labeled cells in both the BSTLS and BSTMS with a slight preponderance in the lateral division.

Case 72R: The injection site was located in the retrorubal ¢eld, with a small amount of uptake in the caudal densocellular region. The BSTL contained the majority of labeled cells, compared to the BSTM which had relatively few labeled cells (Fig. 6D^F). Rostrally, a small cluster of labeled cells in the ventral medial caudate was continuous with labeled cells in the BSTLD, encircling the anterior commissure. Several labeled cells were also seen beneath the ventrolateral putamen (Fig. 6D). Further caudal, there were many labeled cells in the BSTLJ and BSTLC, with a scattered number of labeled cells in the dorsal BSTLcn (Fig. 6E). The BSTLP also contained a moderate number of labeled cells which formed a semicircle around the ventral pallidum. There were occasional labeled cells in the lateral BSTM, and a light distribution of labeled cells in the SLEAm. At caudal levels, there was a cluster of labeled cells in the BSTLS, and scattered labeled cells in the BSTMS (Fig. 6F). In the basal forebrain, labeled cells in the SLEAc continued into the cen-

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Fig. 5. Case 25. (A^C) Distribution of retrogradely labeled cells in the BST/SLEA subdivisions at three rostrocaudal levels. Each dot represents three to four labeled cells. (D,DP) Photomicrographs of near-adjacent sections (boxed area in A) stained for AChE (D) and tracer/Nissl, shown under blue ¢ltered light (DP). Labeled cells in the BSTLD and BSTLJ continue into the ventromedial striatum (arrows). (E) Photomicrograph of HRP-positive cells in the BST. Note the relative paucity of labeled cells in the BSTLcn.

tral nucleus, and a smaller number of labeled cells were seen in the SLEAm. Case 72L (not shown) and 67. These injection sites were located in the lateral SNc at slightly di¡erent ros-

trocaudal levels. In both cases, the majority of labeled cells were seen in the BSTL with relatively few labeled cells in the BSTM. The distribution of labeled cells was similar in both cases. At rostral levels, labeled cells were

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Fig. 6. Case 68R. (A^C) Distribution of retrogradely labeled cells in the BST/SLEA subdivisions at three rostrocaudal levels. Case 72R. (D^F) Distribution of retrogradely labeled cells in the BST/SLEA subdivisions at three rostrocaudal levels. In each case, a dot represents three to four labeled cells.

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Fig. 7. Case 67. (A^C) Distribution of retrogradely labeled cells in the BST/SLEA subdivisions at three rostrocaudal levels. Each dot represents three to four labeled cells. (B) Arrow points to labeled cells in the SLEAc, some of which diverge and enter the SLEAm. (C) Arrow shows labeled cells in the SLEAc which continue into the amygdalostriatal area caudally. (D) Photomicrograph of a section double labeled for HRP and CaBP in the region of the NBM. Most HRP-positive cells (arrows) are medium-sized. Scale bar = 100 Wm.

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Fig. 8. Case 48. (A^C) Distribution of retrogradely labeled cells in the BST/SLEA subdivisions at three rostrocaudal levels. Each dot represents three to four labeled cells. (D) Photomicrograph (boxed area in A) showing a Nissl-stained section containing LY-positive cells in the BSTLC, BSTLJ, and BSTLP. Scale bar = 100 Wm.

moderately concentrated in clusters in the BSTLD, with many labeled cells in the BSTLJ (Fig. 7A). Labeled cells continued into the adjacent striatum. More caudally, there was a high concentration of HRP-labeled cells in the BSTLJ and BSTLC, and a moderate concentration in

the BSTLP (Fig. 7B,D). In contrast, there was a light distribution of labeled cells in the dorsal BSTLcn. Labeled cells in the BSTLP descended medially in the SLEAc under the pallidum (Fig. 7B, arrow). The BSTM contained relatively few labeled cells. At caudal

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levels (Fig. 7C), labeled cells were found in the BSTLS which continued into the ventromedial body of the caudate nucleus. Labeled cells were also found in the caudal ventral putamen and appeared to be continuous with a dense concentration of labeled cells in the rostral amygdalostriatal area (Fig. 7C, arrow). Labeled cells in the SLEAc in a region of the NBM were mostly mediumsized, and were dispersed among, but generally did not include, the magnocellular cells (Fig. 7D). There were few to no labeled cells in the BSTMS, and a light distribution of HRP-positive cells in the SLEAm. Injection sites centered in the densocellular cells (ventral tier) Case 48: Labeled cells throughout the extended amygdala were less densely concentrated compared to cases in which injection sites were located within the dorsal tier. In contrast to cases with dorsal tier injection sites, there were only scattered labeled cells in the BSTLD, BSTLJ and BSTLC rostrally. The majority of labeled cells were in the BSTLP. There were few to no labeled cells in the BSTLcn, and no labeled cells in the BSTM (Fig. 8A, D). Caudal to the anterior commissure scattered labeled cells were seen in the anterior SLEAc beneath the internal capsule (Fig. 8B). Retrogradely labeled cells were also lightly dispersed beneath the posterior limb of the anterior commissure and among the cells of the NBM. Few to no labeled cells were seen in the SLEAm. At caudalmost levels, LY-positive cells were seen in the SLEAc as it entered the CeN (Fig. 8C). There were few to no labeled cells in the SLEAm, BSTLS, or BSTMS. Case 72LY: This injection site was caudal to the injection site in case 48, and similarly centered in the densocellular region. Similar to case 48, there were relatively few retrogradely labeled cells in the BSTLD, BSTLJ, or BSTLC rostrally compared to cases containing dorsal tier injection sites (Fig. 9A, B). The BSTLP had a moderate number of labeled cells (Fig. 9B), and the BSTLcn contained a light concentration of labeled cells. There were scattered labeled cells in the BSTLS with few to none in the BSTMS (Fig. 9C). A light to moderate distribution of labeled cells was found in the lateral SLEAc, with a moderate number of LY-positive cells, some of which intermingled with the magnocellular cells of the NBM, found caudally. These labeled cells formed a continuous stream into the medial subdivision of the central nucleus (data not shown). Scattered LY-positive cells were seen in the SLEAm. Injection sites centered in the cell columns (ventral tier/pars reticulata) Case 62 (not shown): This injection site resulted in scattered labeled cells in the BSTLD and adjacent caudate nucleus. However, there were few to no cells in the BSTLcn, BSTLJ, BSTLP. Scattered labeled cells were seen in anterior dorsal SLEA. Cases 68L and 69 (not shown): The injection sites were in the central, and lateral thirds of the pars reticulata/cell columns, respectively. There were no

Fig. 9. Case 72LY. (A^C) Distribution of retrogradely labeled cells in the BST/SLEA subdivisions at three rostrocaudal levels. Each dot represents three to four labeled cells.

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retrogradely labeled cells in the BSTL or dorsal SLEA. Anterograde cases Case 39 (Fig. 10): This injection site was located in the BSTLP. Labeled ¢bers were found throughout the SNc, mainly in the dorsal tier, but extending into the dorsal densocellular region. At rostral levels, labeled ¢bers were concentrated in the medial dorsal SNc (Fig. 10A). A light concentration of labeled ¢bers extended into the medial densocellular region. From the medial SNc, a moderate concentration of labeled ¢bers traveled horizontally across the entire mediolateral extent of the dorsal tier, giving o¡ ¢ne branching ¢bers. At rostrocentral levels of the midbrain (Fig. 10B, E), labeled ¢bers were concentrated in the central dorsal tier with some labeled ¢bers extending into the dorsal densocellular area. At more caudal levels, few labeled ¢bers were seen in the caudal VTA, but there was a moderate distribution of labeled ¢bers in the lateral dorsal tier (Fig. 10C). At the most caudal levels of the midbrain, labeled ¢bers were concentrated mainly in the lateral half of the SNc (Fig. 10D). In particular the retrorubal ¢eld contained a moderate to high concentration of labeled ¢bers. Some thin varicose labeled ¢bers entered the densocellular region ventral to the retrorubal ¢eld. At all rostrocaudal levels, there were few thin, varicose labeled ¢bers in the cell columns and pars reticulata. Thick, tortuous labeled ¢bers relatively devoid of varicosities were scattered throughout the SNc (not illustrated), and were interpreted to be ¢bers of passage. Case 42 (Fig. 11): The injection site in this case was located in the posterior SLEAc. Labeled ¢bers extended dorsally and ventrally around the pallidum, terminating in the BSTLP and medial CeN. There were few to no labeled ¢bers over the BSTM or medial amygdaloid nucleus (Fig. 11F). In the midbrain, anterogradely labeled ¢bers were distributed across the mediolateral extent of the SNc at all rostrocaudal levels (Fig. 11A^ D). The preponderance of labeled ¢bers was in the dorsal tier, with some labeled ¢bers extending ventrally into the densocellular region. Labeled ¢bers in these two regions were mostly ¢ne caliber and branching, and contained numerous varicosities (Fig. 11E). In contrast, there were few ¢ne branching ¢bers in the cell columns or pars reticulata. At rostrocentral levels (Fig. 11A, B), labeled ¢bers were mainly found in the dorsal tier, with a few labeled ¢bers extending into the dorsal densocellular region. Labeled ¢bers were more densely distributed caudally, with the caudal VTA and retrorubal ¢eld having high densities of labeled ¢bers (Fig. 11C, D). However, a moderate concentration of labeled ¢bers was also seen in the dorsal densocellular region. At the most caudal levels of the midbrain, labeled ¢bers were concentrated predominantly over the retrorubal ¢eld and VTA (Fig. 11D). However, a few beaded labeled ¢bers extended ventrally into the densocellular region. Scattered thick and `corkscrew'-like labeled ¢bers with no varicosities were seen throughout the SNc and SNr (data not shown) and interpreted as ¢bers of passage.

819 DISCUSSION

BSTL and BSTM inputs to the DA neurons Both the BSTL and BSTM project mainly to the DA cells in the dorsal SNc: the dorsal tier receives the strongest inputs, the densocellular region receives a moderate input, and the cell columns/pars reticulata receive relatively little input. In support of these ¢ndings, previous anterograde and retrograde studies in rats and cats show that the dorsal SNc is the main recipient of BST a¡erents (Conrad and Pfa¡, 1976; Meibach and Siegel, 1977; Phillipson, 1979; Swanson, 1979; Holstege et al., 1985). In addition, the present study shows that the primate BSTL has a relatively stronger input to the DA neurons compared to the BSTM. This is demonstrated by the fact that all injections into the dorsal SNc resulted in a relatively greater concentration of labeled cells in the BSTL than in the BSTM. This di¡erential distribution suggests that the BSTL plays a relatively larger role in a¡erent regulation of the DA system. Our results also demonstrate that BSTL and BSTM inputs are di¡erentially organized, consistent with the developmental and functional di¡erences of these structures (Johnston, 1923; Bayer, 1987; Alheid and Heimer, 1988). The BSTL and its continuation into the SLEAc in£uences a broad mediolateral extent of the dorsal tier and densocellular region of the ventral tier, resembling CeN a¡erents (Price and Amaral, 1981; Fudge and Haber, 2000; Gonzales and Chesselet, 1990; Vankova et al., 1992). However, within this broad projection the BSTL subdivisions show di¡erences in their inputs to the DA subpopulations. The BSTLcn is set apart from the other BSTL subdivisions by its paucity of nigral inputs. In this sense, the BSTLcn projection resembles the restricted projection of its caudal counterpart, the CeN, lateral subdivision (CeLcn) (Fudge and Haber, 2000). In marked contrast to the BSTLcn, the BSTL subdivisions surrounding the BSTLcn ^ namely, the BSTLD, BSTLC, and BSTLJ ^ are densely labeled following injections into the dorsal tier. The BSTLD projects mainly to the region of the VTA, while the BSTLJ and BSTLC project across the mediolateral extent of the dorsal tier. Labeled cells in the BSTLJ were particularly densely concentrated after injections into the caudal dorsal tier, including the retrorubal ¢eld (cases 72R, 67, and 72L). Strong BSTLJ inputs to the caudolateral SNc and retrorubal ¢eld have also been shown using anterograde tracers in the rodent (Dong et al., 2000). In contrast to the more anteriorly situated nuclei, the BSTLP contains labeled cells following injections into both the dorsal tier and densocellular region of the ventral tier. However, the concentration of labeled neurons in the BSTLP is signi¢cantly less following injection sites centered in the densocellular area, indicating that while the BSTLP projects to both DA subpopulations, there is a greater in£uence of the dorsal tier. Anterograde results (case 39, Fig. 10) support this conclusion with labeled axon terminals concentrated mainly over the dorsal tier but extending ventrally into the densocellular area. Retrograde injection sites that back-labeled cells in the

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Fig. 10. Case 39. (A^D) Distribution of labeled ¢bers at four rostrocaudal levels through the ventral midbrain after an injection in the BSTLP. Light gray region indicates the ventral tier. (E) High power bright-¢eld photomicrograph of a section double labeled for tracer and TH (boxed area in B). Labeled ¢bers containing varicosities are found in proximity to TH-positive cells. Scale bar = 50 Wm.

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Fig. 11. Case 42. (A^D) Distribution of labeled ¢bers at four rostrocaudal levels through the ventral midbrain after an injection in the SLEAc. Light gray region indicates the ventral tier. (E) High power photomicrograph of anterogradely labeled ¢bers in the ventral midbrain (boxed area in C). (F) Dark-¢eld photomicrograph of the injection site. Anterogradely labeled ¢bers stream into the BSTLP dorsally, and into the medial central nucleus ventrally. Scale bar = 50 Wm.

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BSTLP also resulted in a near-continuous stream of labeled cells in the SLEAc and medial CeN, supporting the continuity of these structures. While the SLEAc alone projected to the central and lateral SNc, inputs from both the SLEAc and SLEAm converged in the medial SNc. This convergence indicates that the medial SNc is uniquely in£uenced by elements of both the central and medial extended amygdala. Converging SLEAc and SLEAm inputs in the region of the VTA have also been shown in the rat (Grove, 1988a). The BSTM and its continuum with the SLEAm have the densest inputs to the medial SNc, and relatively fewer inputs to the central and lateral SNc. Labeled cells in the BSTM form a near-continuous stream with labeled cells in the SLEAm which £ow ventrally then laterally along the base of the brain into the medial amygdaloid nucleus (cases 25 and 68). The BSTM/SLEAm-nigral projection resembles the organization of nigral a¡erents from the medial amygdaloid nucleus (Canteras et al., 1995) which forms the caudal end of the medial `extended amygdala'. The BSTL subdivisions Much of the BSTL (including the BSTLD, the BSTLJ, BSTLC, and BSTLcn) is composed of medium spiny neurons (McDonald, 1982; McDonald, 1983; Cassell and Gray, 1989; Sun and Cassell, 1993). Despite this, the BSTL subdivisions are distinguished by a marked histochemical heterogeneity (Gaspar et al., 1985; Gaspar et al., 1987; Lesur et al., 1989; Martin et al., 1991; Walter et al., 1991; Kaufmann et al., 1997; Heimer et al., 1999 and present results), suggesting di¡erential input/output pathways. A¡erents to the BSTL generally derive from many of the same cortical, thalamic, amygdaloid, hippocampal, hypothalamic, and brainstem regions that innervate the ventral striatum (Krettek and Price, 1978; Weller and Smith, 1982; Russchen et al., 1985a; Russchen et al., 1985b; Grove, 1988b; McDonald, 1991b). However, the speci¢c inputs and outputs of the individual BSTL subdivisions are only now beginning to be elucidated (Moga et al., 1989; Sun et al., 1991; Alden et al., 1994; Canteras et al., 1994; Kozicz et al., 1998; McDonald et al., 1999; Dong et al., 2000). The BSTLcn stands out in having a speci¢c subset of connections which resemble those of the CeLcn. A¡erents to both are derived from the posterior insula (McDonald and Jackson, 1987; Yasui et al., 1991; Sun et al., 1994) and lateral parabrachial nucleus, (Saper and Loewy, 1980; Bernard et al., 1993; Alden et al., 1994) and e¡erents project to the lateral parabrachial nucleus (Moga et al., 1989; Moga et al., 1990; Petrovich and Swanson, 1997), central gray (Gray and Magnuson, 1992), dorsal vagus (Gray and Magnuson, 1987), and the BSTLP/SLEAc (Grove, 1988a; Sun et al., 1991; Sun and Cassell, 1993). These pathways suggest that the BSTLcn and CeLcn mediate autonomic responses to pain and stress(Bernard et al., 1994; Bernard et al., 1996). Another way the BSTLcn and CeLcn stand out is in their limited inputs to SNc, indicating relatively little direct in£uence on the DA cells (present results and Fudge and Haber, 2000).

In contrast, the BSTLD, BSTLJ, and BLTLC subdivisions receive few of the lateral parabrachial inputs characteristic of the BSTLcn (Alden et al., 1994). A¡erent projections originate in the central and basolateral amygdala, agranular and dysgranular insula, and piriform cortex (Krettek and Price, 1978; McDonald et al., 1999). The medial parabrachial nucleus, which relays gustatory cues, projects to the BSTLC and BSTLD (Alden et al., 1994). Collectively, these inputs relay emotionally relevant and gustatory information to the BSTLD/BSTLJ/BSTLC, and are similar to a¡erent projections innervating the ventral striatal shell (McDonald, 1991a). The present study shows that the BSTLJ/BSTLC/ BSTLD also resembles the shell with respect to selective outputs to the dorsal tier (Lynd-Balta and Haber, 1994c). Therefore, the BSTLD/BSTLJ/BSTLC and shell are similar not only in their a¡erent innervation, but also in their e¡erent projection to the dorsal tier neurons. The BSTLP, like the medial CeN, contains heterogeneous neurons (McDonald, 1983; Sun and Cassell, 1993). The BSTLP receives inputs from the prelimbic and olfactory cortex, amygdala, medial parabrachial nucleus, and pontine taste centers (Norgren, 1976; Krettek and Price, 1978; Weller and Smith, 1982; McDonald, 1991b; Alden et al., 1994; McDonald et al., 1999), in addition to prominent `intrinsic' a¡erents from the BSTLcn, CeLcn, and SLEAc, proposed to relay noxious or painful cues (Henke, 1984; Rao et al., 1987; Han and Ju, 1990; Sun et al., 1991; Sun and Cassell, 1993). The BSTLP thus combines appetitive and aversive qualities of stimuli relayed from cortical, subcortical, and brainstem centers. We found that the BSTLP/SLEAc is in a position to channel this information to broadly in£uence the dorsal tier and densocellular neurons, similar to the medial CeN-nigral pathways (Fudge and Haber, 2000) (Fig. 12). The concept of the `extended amygdala' The extended amygdala is conceptualized as a neuronal continuum between the centromedial amygdala and the BST which is divided into central and medial divisions (DeOlmos and Ingram, 1972; Alheid and Heimer, 1988). Similarities between the BST and centromedial amygdala have been documented in histochemical, cytoarchitectural, and tracing studies (DeOlmos and Ingram, 1972; McDonald, 1982; Schwaber et al., 1982; McDonald, 1983; Grove, 1988a; Grove, 1988b; Alheid et al., 1995; Heimer et al., 1999; Shammah-Lagnado et al., 1999). However, the concept of the `extended amygdala' remains controversial, with some authors proposing that the centromedial amygdala is more `striatal-like', and not part of a separate structure (Swanson and Petrovich, 1998; Cassell et al., 1999). The present study supports the concept of the extended amygdala in several ways. Retrograde tracer injections into the dorsal SNc result in two largely separate columns of labeled cells. These columns are organized in dorsolateral and medioventral streams which overlap with the histochemically identi¢ed regions of the central and medial extended amygdala, respectively (Martin et al., 1991; Heimer et

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823

Fig. 12. Summary diagram of central extended amygdala inputs to the DA subpopulations. The CeLc/Astr area, BSTLJ, BSTLD (not shown) and BSTLC project preferentially to the dorsal tier neurons. The CeM, SLEAc, and BSTLP have broad inputs to the dorsal tier and densocellular portion of the ventral tier. The BSTLcn and CeLcn have relatively limited inputs. Schematic based on present results and previous work (Fudge and Haber, 2000).

al., 1999). Furthermore, after each injection, labeled cells are found throughout the rostrocaudal extent of the extended amygdala: from the BST through the SLEA and into the centromedial amygdala. These ¢ndings support the concept of the extended amygdala as a uni¢ed rostrocaudal structure with distinct lateral and medial components. Another major ¢nding is that while much of the BSTL contains medium spiny neurons, only speci¢c subdivisions have `striatal-like' e¡erents to the DA subpopulations. The lateral-most BSTL subdivisions ^ the BSTLD/ BSTLJ/BSTLC ^ project selectively to the dorsal tier, speci¢cally resembling the DA projections of the ventral striatum (Lynd-Balta and Haber, 1994c). This is not the case with the BSTLcn, which has few inputs to the DA cells. Our previous results in the caudal extended amygdala mirror this ¢nding. The lateral-most CeN subdivisions (CeN, capsular subdivision (CeLc), and amygdalostriatal area) selectively project to the dorsal tier, while the CeLcn has a restricted input (Fudge and

Haber, 2000). We therefore conclude that only the lateral-most subdivisions of the central extended amygdala can be considered `striatal-like' based on e¡erents to the DA neurons (Fig. 12). Functional implications: BST in motivated responses Earlier studies showing that the BSTL projects to the DA cells suggested a potential pathway by which stressrelated stimuli might activate the DA system (Henke, 1984; Morrell et al., 1984; Holstege et al., 1985; Dunn, 1987; Grove, 1988a; Casada and Dafny, 1991; Gray, 1993; Menzaghi et al., 1993; Bernard et al., 1994). Although the DA system is classically associated with reward (Schultz et al., 1993; Ikemoto et al., 1997; Schultz, 1997), goal-directed behaviors can be altered by adding a stressful component (Ghiglieri et al., 1997; Phillips and Barr, 1997; Bowers et al., 1999; CalvoTorrent et al., 1999). This indicates that ¢nal behavioral outcomes result from interactions between stressful and

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rewarding stimuli. The BSTL may be involved in processing both stress- and reward-related stimuli, which in turn in£uence goal-directed behaviors through the DA system. A major ¢nding of the present study is that the BSTL subdivisions are di¡erentially organized with respect to projections to the DA system. The BSTLcn, like the CeLcn, stands out not only in speci¢c connections to the caudal brainstem, but also in a restricted input to the DA neurons. This organization suggests that the BSTLcn and CeLcn mediate internal stress responses, and that modulation of the DA system is mainly via other pathways. In contrast, the lateral-most subregions of both the BSTL and CeN (the BSTLD/BSTLJ/BSTLC and CeLc/amygdalostriatal areas, respectively) have selective inputs to the dorsal tier neurons, similar to the ventral striatal shell (Lynd-Balta and Haber, 1994c). The dorsal tier neurons project back to the shell, prefrontal cortex, and amygdala (Fallon, 1978; Porrino and Goldman-Rakic, 1982; Deutch et al., 1988; Gaspar et al., 1992; Lynd-Balta and Haber,

1994a), which are involved in exploratory and motivated responses (Cador et al., 1989; Ledoux, 1992; Gallagher and Holland, 1994; Richardson and Gratton, 1996; Robbins and Everitt, 1996; Rebec et al., 1997b; Lee et al., 1998; Richardson and Gratton, 1998). The BSTLP and SLEAc, like the medial CeN, receive the bulk of intrinsic inputs from the central extended amygdala, as well as broad inputs from cortical and subcortical regions. Similarly, the BSTLP/SLEAc (like the medial CeN) has inputs to a wide extent of the dorsal tier and densocellular neurons. This organization indicates that the BSTLP/SLEAc/medial CeN continuum combines information from the internal and external environment to broadly modulate DA outputs not only in `limbic' circuits but also in striatal territories involved in initiating thought and movement (Haber et al., 2000). AcknowledgementsöThis work was supported by MH45573 and The Lucille P. Markey Charitable Trust (S.H.), and the Leonard F. Salzman Fund (J.F.). The authors thank Ms. Evelyn Galban for her valuable technical assistance.

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