THE JOURNAL OF COMPARATIVE NEUROLOGY 295624-661 (1990)

Organization of Cortical, Basal Forebrain, and Hypothalamic Afferents to the Parabrachial Nucleus in the Rat MARGARET M. MOGA, HORST HERBERT, KAREN M. HURLEY, YUKIHIKO YASUI, THACKERY S. GRAY, AND CLIFFORD B. S U E R Departments of Pharmacological and Physiological Sciences and Neurology, Committee on Neurobiology, University of Chicago, Chicago, Illinois 60637 (M.M.M., H.H., K.M.H., Y.Y., C.B.S.); Department of Anatomy, Loyola University Stritch School of Medicine, Maywood, Illinois 60153 (T.S.G.)

ABSTRACT In a previous study (Herbert et al., J. Comp. Neurol. [1990];293:540-580), we demonstrated that the ascending afferent projections from the medulla to the parabrachial nucleus (PB) mark out functionally specific terminal domains within the PB. In this study, we examine the organization of the forebrain afferents to the PB. The PB was found to receive afferents from the infralimbic, the lateral prefrontal, and the insular cortical areas; the dorsomedial, the ventromedial, the median preoptic, and the paraventricular hypothalamic nuclei; the dorsal, the retrochiasmatic, and the lateral hypothalamic areas; the central nucleus of the amygdala; the substantia innominata; and the bed nucleus of the stria terminalis. In general, forebrain areas tend to innervate the same PB subnuclei from which they receive their input. Three major patterns of afferent termination were noted in the PB; these corresponded to the three primary sources of forebrain input to the PB: the cerebral cortex, the hypothalamus, and the basal forebrain. Hypothalamic afferents innervate predominantly rostra1 portions of the PB, particularly the central lateral and dorsal lateral subnuclei. The basal forebrain projection to the PB ends densely in the external lateral and waist subnuclei. Cortical afferents terminate most heavily in the caudal half of the PB, particularly in the ventral lateral and medial subnuclei. In addition, considerable topography organization was found within the individual projections. For example, tuberal lateral hypothalamic neurons project heavily to the central lateral subnucleus and lightly to the waist area; in contrast, caudal lateral hypothalamic neurons send a moderately heavy projection to both the central lateral and waist subnuclei. Our results show that the forebrain afferents of the PB are topographically organized. These topographical differences may provide a substrate for the diversity of visceral functions associated with the PB. Key words: amygdala, hypothalamus, cortex, pons, autonomic nervous system

The parabrachial nucleus (PB) has historically been associated with the central gustatory pathways (Herrick, '05, '48; Barnard, '36; Norgren and Leonard, '73). More recently, the PB has been implicated in a variety of visceral functions, including the regulation of fluid balance (Ohman and Johnson, '86), micturition (Lumb and Morrison, '87), cerebral blood flow (Mraovitch et al., '85), and blood pressure (Mraovitch et al., '82) as well as the modulation of respiration (Cohen, '71; Feldman and Gautier, '76; von Euler et al., '76) and nociception (Hammond and Proudfit, '80; Brennan et al., '87; Girardot et al., '87). This functional diversity is reflected in the anatomical complexity of the PB. The PB contains ten cytologically distinct subnuclei, and each PB efferent projection arises from a characteristic neuronal population composed of one or more subnuclei o 1990 WILEY-LISS, INC.

(Fulwiler and Saper, '84). The parabrachial afferent connections may also be topographically organized (Saper, '82; Cechetto et al., '85; Shapiro and Miselis, '85), but there has been no comprehensive study to address this issue. In a previous report (Herbert et al., 'go), we demonstrated that the ascending afferent projections from the medulla mark out characteristic terminal domains within the PB. In the present study, we examine both the origin and the pattern of termination of the forebrain afferents to the PB by using anterograde and retrograde tracers. A preliminary account of this study was previously reported (Moga et al., '86).

Accepted December 13,1989.

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PARABRACHIAL FOREBRAIN AFFERENTS Abbreviations AA ac Ace ACo AHA A1 AON ARC AV AVPV

B BL BM BST AL AM DI. PI PL PM PO VL VM C cc Ce CeL CeM CG CM CP CP ctf CUN DB DI DMH DR DT ec En EW FF fr fx GI GP I ic ICj ICOl

IL IP La LC LD LDT LGd LGv LH LHb LM lot LP LPO LS LV

MD Me Mes5 MG MgPO MHb ml

anterior amygdaloid area anterior commissure accessory magnocellular nucleus anterior cortical nucleus of the amygdala amygdalohippocampal transition area agranular insular cortex anterior olfactory nucleus arcuate nucleus anteroventral nucleus of the thalamus anteroventral periventricular nucleus Barrington's nucleus basolateral nucleus of the amygdala basomedial nucleus of the amygdala bed nucleus of the stria terminalis and subnuclei anterior lateral subnucleus anterior medial subnucleus dorsal lateral subnucleus posterior intermediate subnucleus posterior lateral subnucleus posterior medial subnucleus preoptic subnucleus ventral lateral subnucleus ventral medial subnucleus claustrum corpus callosum central nucleus of the amygdala central nucleus of the amygdala, lateral subdivision1 central nucleus of the amygdala, medial subdivision central gray centromedian nucleus of the thalamus cerebral peduncle caudate and putamen central tegmental field cuneiform nucleus nucleus of the diagonal band dysgranular insular cortex dorsomedial nucleus of the hypothalamus dorsal raphe nucleus dorsal tegmental nucleus external capsule endopiriform nucleus Edinger-Westphal nucleus fields of Forel fasciculus retrodexus fornix granular insular cortex globus pallidus intercalated nuclei of the amygdala internal capsule islands of Calleja inferior colliculus infralimbic cortex interpeduncular nucleus lateral nucleus of the amygdala locus coeruleus laterodorsal nucleus of the thalamus laterodorsal tegmentd nucleus lateral geniculate, dorsal subnucleus lateral geniculate, ventral subnucleus lateral bypothalamus lateral habenular nucleus lateral mammillary nucleus lateral olfactory tract lateroposterior nucleus of the thalamus lateral preoptic area lateral septum lateral ventricle mediodorsal nucleus of the thalamus medial nucleus of the amygdala mesencephalic nucleus and tract of the trigeminal nerve medial geniculate nucleus magnocellular preoptic nucleus medial habenular nucleus medial lemniseus

mlf MM MnPO MPN Mo5 mt n4 N4 NAc NLOT ot PBG PBI cl dl el ex1 il KF sl vl PBm exm med PC PD

PF PH PIR PLCO PMCo PPT PRh PS PS5

PT PTA PVH aP dp IP mp Pm Pt' PVT Rch Re Rh RN RP RRF Rt SCh SCOl SCP SCR SFO SHY

SI

sm SNc SNr

so st

STb su5 SUM Tu TT VL VMH VPM vst ZI

medial longitudinal fasciculus medial mammillary nucleus median preoptic nucleus medial preoptic nucleus motor nucleus of the trigeminal nerve mammillothalamic tract trochlear nerve nucleus of the trochlear nerve nucleus accumbens nucleus of the lateral olfactory tract optic tract parabigeminal nucleus parabrachial nucleus, lateral subnuclei central lateral subnucleus dorsal lateral subnucleus external lateral subnucleus extreme lateral subnucleus internal lateral subnucleus Kolliker-Fuse subnucleus superior lateral subnucleus ventral lateral subnucleus parabrachial nucleus, medial subnuclei external medial subnucleus medial subnucleus posterior commissure posterodorsal nucleus parafascicular nucleus of the thalamus posterior hypothalamic area piriform cortex posterior lateral cortical nucleus of the amygdala posterior medial cortical nucleus of the amygdala pedunculopontine tegmental nucleus perirhinal cortex parastrial nucleus principal sensory nucleus of the trigeminal nerve paratenial nucleus of the thalamus pretectal area paraventricular nucleus of the hypothalamus and subdivisions anterior parvicellular subdivision dorsal parvicellular subdivision lateral parvicellular subdivision medial parvicellular subdivision posterior magnocellular subdivision periventricular subdivision paraventricular nucleus of the thalamus retrochiasmatic area nucleus reuniens rhomboid nucleus of the thalamus red nucleus rapbe pontis nucleus retrorubral field reticular nucleus of the thalamus suprachiasmatic nucleus of the hypothalamus superior colliculus superior cerebellar peduncle superior central raphe nucleus subfornical organ septohypothalamic nucleus substantia innominata stria medullaris substantia nigra pars compacta substantia nigra pars reticulata supraoptic nucleus of the hypothalamus stria terminalis subthalamic nucleus supratrigeminal nucleus supramammillary nucleus olfactory tubercle tenia tecta ventrolateral nucleus of the thalamus ventromedial nucleus of the hypothalamus ventral posteromedial nucleus of the thalamus ventral spinocerebellar tract zona incerta

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MATERIALS AND METHODS Adult male albino rats, weighing 250-350 g, were anesthetized with 7% chloral hydrate.

Wheat germ agglutinin-horseradish peroxidase (WGA-HRP)experiments Injections (3-50 nl) of 1% WGA-HRP were made into the parabrachial nucleus and into nuclei with projections to the PB by using an air pressure injection system (Amaral and Price, ’83). Injections were made over a 10-15 minute period, and the pipette was left in place for an additional 10 minutes. After 48 hours of survival, the animals were reanesthetized and perfused through the ascending aorta with 200 ml of saline followed by 500 ml of a solution of 0.5% paraformaldehyde-1.25% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The brains were removed and soaked overnight in a 30% sucrose solution in phosphate-buffered saline (PBS),pH 7.4, at 4OC. Fifty micrometer sections were cut on a freezing microtome and processed according to the tetramethylbenzidine (TMB) method of de Olmos et al. (’78). Sections were mounted on glass slides, dried overnight, counterstained with thionin, rapidly dehydrated in a series of graded alcohols at 4’C, cleared with xylene, and then coverslipped with Histoclad mounting medium. Sections were viewed with darkfield polarization optics. The locations of the labeled neurons and fibers were mapped on camera lucida drawings of the sections. We defined the border of an injection site on the basis of our previous experience with a series of multiple, closely spaced injections (Saper, ’82; Saper and Levisohn, ’83), as the area of uniform dense reaction product surrounding the pipette tip. A neuron was considered positive for WGA-HRP labeling if fine granular TMB precipitate could be distinguished within the cytoplasm and extending at least partially into the proximal dendrites.

Phaseolus vulgaris leukoagglutinin (PHA-L) experiments A 2.5% solution (0.01 M phosphate buffer, pH 7.5) of PHA-L (Vector Laboratories L-1110) was placed in glass micropipettes with a tip diameter of 25-50 pm. This solution was iontophoresed into PB afferent cell populations by using 5-8 pA of positive direct current in 7 second pulses for 30 minutes. After 7-21 days of survival, the animals were deeply reanesthetized with 7 % chloral hydrate and perfused through the ascending aorta with 200 ml of saline followed by 500 ml of 4 % paraformaldehyde-0.2% glutaraldehyde in a 0.1 M phosphate buffer, pH 7.4, and then followed by 30% sucrose in PBS. The brains were removed and cut into 30-40 pm sections on a freezing microtome. A one-in-three series of sections was processed according to a modification of the PHA-L method described by Gerfen and Sawchenko (’84). Briefly, sections were treated with 0.04% hydrogen peroxide in PBS with 0.25% Triton-X, preincubated in either 3% normal swine or donkey serum diluted in PBS with 0.25% Triton-X, and then incubated in the primary antibody (goat anti-PHA [E + L], Vector Laboratories AS-2224, diluted 1:1,000 in the preincubation solution) for 24 hours at 4°C. Next, the sections were rinsed in PBS, incubated for 1hour in either a HRP-labeled secondary antibody (swine antigoat IgG, Tag0 6431, diluted 150) or a biotinylated secondary antibody followed by 1 hour in an avidin-conjugated HRP complex (Bethesda Research Laboratories, biotinylated donkey antigoat IgG, 53131,1:200,and streptavidin-HRP conju-

gate, 8KB105, 1:400). Sections were then rinsed in PBS and processed with a solution containing 0.05% 3‘,3’-diaminobenzidine (Sigma) and 0.0170 hydrogen peroxide. Sections were rinsed again, mounted onto gelatin-coated slides, air-dried, dehydrated in graded alcohols, cleared in xylene for 1-2 weeks, and coverslipped with Histoclad mounting medium. Sections were viewed with both darkfield and brightfield microscopy.

RESULTS Cytoarchitecture of the parabrachial subnuclei In an earlier study, Fulwiler and Saper (’84) divided the

PB complex into ten subnuclei based on the size, shape, and staining characteristics of the PB neurons and on the locations of the efferent cell populations. Further observation of the afferent and efferent connections of the PB complex has led us to modify slightly the boundaries of some of those subnuclei. In particular, we now define the rostral portion of the ventral lateral subnucleus as extending further medially than previously indicated. At rostral levels, the ventral lateral subnucleus caps the dorsal medial border of the superior cerebellar peduncle, and is traversed by the mesencephalic tract and nucleus of the trigeminal nucleus (e.g., Fig. 7B). Like its caudal part, the rostral part of the ventral lateral subnucleus contains small, ovoid cells, which are distinguishable from the cells in the overlying central lateral subnucleus by their greater packing density. Furthermore, the ventral lateral subnucleus does not extend as far laterally as indicated in Figures 8-10 of Fulwiler and Saper (’84); instead, our tracing studies (see below) indicate that the flattened neurons located along the dorsal lateral surface of the superior cerebellar peduncle are part of the external lateral subnucleus. Fulwiler and Saper (’84) observed that the external lateral subnucleus contains an inner zone that projects to the substantia innominata and an outer zone that projects to the hypothalamus and amygdala. Further inspection revealed subtle cytoarchitectural differences between these two zones. Cells in the lateral part of external lateral subnucleus are slightly larger and more closely packed than the “flattened” cells in the medial part of this subnucleus. In addition, the results of the current study and those of Herbert et al. (’90) indicate that these two zones receive different afferents. However, these differences do not seem sufficient to delineate another subnucleus; thus, we use the terms “outer zone” and “inner zone” in some cases to describe more accurately the labeling within the external lateral subnucleus. The other PB subnuclei were as described previously (Fulwiler and Saper, ’84).

Retrograde labeling experiments Forty-eight animals received injections into the parabrachial nucleus. In five rats, large (approximately 20-30 nl) injections included most of the P B subnuclei, with little involvement of adjacent nuclei. These five experiments showed similar retrograde labeling in the forebrain and midbrain and are represented by case R407, which is described in detail below. A series of small PB injections (approximately 3-6 nl and usually including only one or two subnuclei) were made to examine the afferents to specific PB subnuclei; the results from these experiments will be presented where they supplement or clarify the anterograde

PARABRACHIAL FOREBRAIN AFFERENTS transport experiments described in the next section. A summary diagram of these small injection sites is presented in Figure 27 of Herbert et al. (’90). The injection site in experiment R407 (Fig. 1P) included all of the P B subnuclei (except for the superior lateral subnucleus, the outer zone of the external lateral subnucleus, and most of the Kolliker-Fuse nucleus) and also the supratrigeminal nucleus, located just ventral to the medial subnucleus, the mesencephalic nucleus of the trigeminal nerve, and a small part of the pontine reticular formation. The periaqueductal gray matter, cuneiform nucleus, locus coeruleus, and principal sensory nucleus of the trigeminal nerve were excluded from the injection site. The distributions of the retrogradely labeled cells in the forebrain, midbrain, and pons in case R407 are depicted in Figure 1A-P and described in the following paragraphs. The labeling of neurons in the lower brainstem in experiment R407 is shown in Figure 2 of Herbert et al. (’90). Although there is very little uptake of WGA-HRP by uninjured fibers of passage (Grob et al., ‘82; Gibson et al., ’84),this tracer may be transported by axons that are damaged by the injection pipette. For example, we noted retrogradely labeled neurons in the deep cerebellar nuclei following large injections (including R407) in which the pipette tip penetrated the superior cerebellar peduncle. We did not observe any axonal labeling within the substance of the parabrachial nucleus following a WGA-HRP injection into the deep cerebellar nuclei (Moga and Saper, unpublished observations), suggesting that the retrograde cerebellar labeling following large P B injections was due to uptake by damaged fibers of passage. Hence the retrograde labeling illustrated for experiment R407 was taken as only a first approximation of the parabrachial afferents. These observations were then used as a basis for the anterograde tracing experiments to confirm the distribution of the afferents to the PB (see next section). Cortex. A large number of retrogradely labeled cells was found in layer V of the frontopolar cortex (Fig. IA). With succeeding sections, these cells condensed ventrally into two clusters, one medially in the infralimbic cortex, and the other laterally in the lateral frontal and insular cortices. Labeled cells in the infralimbic cortex were most numerous in layer V of its caudal half (Figs. IC, 2A). In the insular cortex, WGA-HRP-labeled neurons were present in layer V throughout its rostrocaudal extent, particularly in its agranular and dysgranular subdivisions (Figs. 1C-H, 2B). A few labeled cells were found more caudally in the perirhinal cortex (Fig. 11,J). Deep to the insular cortex, many labeled neurons were found in the thin, caudal portion of the claustrum (Fig. 1D-H). Basal forebrain. Retrogradely labeled neurons were particularly numerous in a continuum running from the bed nucleus of the stria terminalis (BST), through the substantia innominata, and into the central nucleus of the amygdala. Within the BST, labeled neurons were most numerous in the dorsal lateral and posterior lateral subnuclei, with many neurons also present in the anterior lateral, ventral lateral, and preoptic subnuclei (Figs. 1E,F, 3A,C; for a description of the BST subnuclei, see Moga et al., ’89). The parastrial nucleus (Simerly et al., ’84) contained a few retrogradely labeled cells (Fig. 1E). Many labeled neurons were present in the sublenticular substantia innominata, anterior amygdaloid area, and magnocellular preoptic nucleus (Figs. 1E-G, 4A), and in each of the subdivisions of the central nucleus of the amygdala (Figs. lH,I, 3B,D). Anterograde labeling was

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particularly heavy in both the lateral and the lateral capsular subdivisions of the central nucleus of the amygdala (Fig. 3B) and in the dorsal lateral subnucleus of the BST. Several intercalated nuclei of the amygdala contained both retrograde and anterograde label (Fig. 4B). In particular, a cluster of labeled cells and fibers was observed in the “beaded” intercalated cell chain located between the central nucleus and the basolateral and lateral amygdaloid nuclei (Fig. 4B; for a description of the intercalated nuclei, see Millhouse, ’86). A labeled intercalated nucleus, previously described by Moga and Gray (’%a), was found between the intermediate and external fiher capsules, just rostral to the basolateral amygdaloid nucleus and ventrolateral to both the central nucleus and the posterior limb of the anterior commissure. Labeling was also observed in an intercalated nucleus located just dorsal to the rostral pole of the central nucleus beneath the striatum and in a few small intercalated nuclei located ventral and dorsal to the posterior limb of the anterior commissure. Hypothalamus. In the hypothalamus, retrogradely labeled neurons were numerous in the median preoptic nucleus (Fig. 1E). A small number of labeled neurons was located in the anteroventral periventricular nucleus (Fig. 1E,F), and in the medial preoptic nucleus, primarily along its lateral border (Fig. 1F). Labeled neurons were more numerous in the lateral preoptic area (Fig. 1F) and in the paraventricular nucleus, particularly within its medial and lateral parvicellular subdivisions (Figs. l H , 5A-C). Scattered retrogradely labeled neurons were found within the retrochiasmatic area (Fig. IH); these neurons extended caudally into the ventrolateral portion of the ventromedial nucleus (Figs. 11)and into the arcuate nucleus (Fig. 11-K). A dense cluster of labeled neurons was present in the tuberal lateral hypothalamus and the adjacent zona incerta between the fornix and the internal capsule (Figs. lI,J, 6A,C). With succeeding sections, this cluster appeared to split into two groups, one adjacent to the internal capsule and the other surrounding the fornix (Fig. 11-K). The group surrounding the fornix appeared continuous caudally with labeled cells in the dorsal hypothalamic area (Fig. 1J). Retrogradely labeled cells in the lateral hypothalamus adjacent to the internal capsule formed a “cap” along the medial edge of the subthalamic nucleus; these cells extended to the caudalmost limits of the lateral hypothalamus (Figs. IK, 6B,D). Laheled cells within the dorsomedial hypothalamic nucleus were restricted to its large-celled, loosely packed portion (Fig. 1J). Only scattered cells were labeled in the posterior hypothalamic area (Fig. 1K). Midbrain and pons. In the midbrain, retrograde and axonal labeling was extensive in the central gray substance, particularly in its ventrolateral quadrant (Fig. 1L-0). Retrogradely labeled neurons were also present along the dorsal surface of the compact zone of the substantia nigra; with succeeding sections, these neurons became concentrated in the lateral part of this zone (Fig. lL,M). Many labeled neurons were observed surrounding the central tegmental field bilaterally (Fig. 1M). More caudally, a dense cluster of retrograde and axonal labeling was found in the retrorubral field (Fig. 1N; for a description of this nucleus, see Rye et al., ’87). A few labeled neurons were seen in the pedunculopontine nucleus, the dorsal raphe nucleus, the EdingerWestphal nucleus, and the superior central raphe nucleus (Fig. 1M-0). In the pons, many retrogradely labeled neurons were found in the contralateral parabrachial nucleus, particularly

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Figure 1A-C

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D

Fig. 1. A series of camera lucida drawings illustrating the distribution of retrogradely labeled cells in the Each forebrain and upper brainstem after a WGA-HRPinjection (R407) into the parabrachial nucleus (P). dot represents one retrogradely labeled cell in a single 50-rm-thick section except in the Ce and BST, where each dot represents approximately two cells.

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Figure 1F-G

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Figure 1J-K

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Figure 1 L M

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Figure IN-P

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Fig. 2. Polarization photomicrographs of retrogradely labeled cells in the infralimbic cortex (A), and the insular cortex (B),after a WGA-HRP injection (R407)into the parabrachial nucleus. Bar = 200 pm.

in the ventral lateral and central lateral and Kolliker-Fuse subnuclei (Fig. 1P). Labeled neurons in the mesencephalic nucleus of the trigeminal nerve and the supratrigeminal nucleus were probably a result of the injection site involving the supratrigeminal nucleus (Rokx et al., ’86a,b), in that these were not seen following smaller injections confined to the PB.

Anterograde labeling experiments In this series of experiments, we injected ten of the most prominent forebrain sources of afferents to the PB with WGA-HRP to determine their terminal distributions within the PB. Each of these injections resulted in fine granular labeling in the PB neuropil as well as cellular labeling. The latter can be confidently interpreted as retrograde labeling, but the former requires some clarification. Following injections into sites, such as the thalamus, that receive PB afferents but do not project back to the PB, we found extensive cellular labeling but little if any labeling over the neuropil. Thus we consider it unlikely that the fine granular

label is dendritic. On the other hand, even if the neuropil labeling is considered to be axonal, it could represent axon collaterals of retrogradely labeled PB neurons as well as anterogradely labeled descending fibers (Takeuchi et al., ’85). For this reason, and as an aid for future interpretation, the retrogradely labeled neurons observed in these experiments were included in our line figures. More importantly, we have supplemented our WGA-HRP observations with injections of PHA-L, a lectin tracer that is transported largely in an anterograde direction (Gerfen and Sawchenko, ’84) into seven selected sites. In each case, the pattern of terminal labeling was identical to the WGA-HRP material. Thus, although the WGA-HRP fiber labeling is probably “anterograde” rather than “axonal,” it is important to recognize that, in cases when PHA-L experiments have not been performed, the distribution of this labeling may overestimate the extent of the descending terminal field. litfralirnbic cortex. Nine animals received WGAHRP injections into the infralimbic cortex. Five injections were large (15-20 nl) and included most of the infralimbic

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Fig. 3. Polarization photomicrographs of retrograde and axonal labeling in the caudal part of the bed nucleus of the stria terminalis (BST) (A), and in the central nucleus of the amygdala ( C e ) (B) after a WGA-HRP injection (R407) into the parabrachial nucleus. Bar = 200

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(C,D) illustrate retrogradely labeled cells present in, respectively, A and B. For orientation, the arrows in A and C, and B and D, indicate the same labeled neuron. Bar = 100 Gm.

pm. The high-power, brightfield photomicrographs

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Fig. 4. Photomicrographs of retrogradely labeled cells in the magnocellular preoptic nucleus (A), and an intercalated nucleus of the amygdala (B).Bar = 100 pm.

cortex. Of the other four injections (6-10 nl), two were centered in the rostral half, one was placed in the caudal half, and the fourth involved primarily the ventral portion of the infralimbic cortex at a midrostrocaudal level. The pattern of anterograde labeling within the PR was identical for each case. Labeling was heaviest after large injections located in mid- to caudal levels of the infralimbic cortex. A representative experiment, case R218, is shown in Figure 7. In this experiment, moderately light fiber labeling was seen throughout the rostral P B (Fig. 7B) with a slight concentration of fibers in the central lateral, extreme lateral, and medial subnuclei. At midlevels of the PB, axonal labeling was moderately dense within the central lateral subnucleus as well as in the medial portion of the medial subnucleus (Fig. 7C). Labeling within the external lateral subnucleus was heavy only a t caudal levels of the PB (Fig. 7D). The internal lateral and, to a lesser extent, the superior lateral and ventral lateral subnuclei were conspicuous by their lack of fiber labeling (Fig. 7C,D).

Four PHA-L injections were placed in the infralimbic cortex. In all four cases, the pattern of anterograde labeling in the PB was identical to that seen after the WGA-HRP experiments. In experiment R652, labeled terminals with boutons were observed in all of the P B subnuclei except for the internal lateral and superior lateral subnuclei, which were devoid of labeling. In summary, the infralimbic projection to the P B terminates in a diffuse pattern, largely avoiding the internal lateral, and to a lesser extent the superior lateral and ventral lateral subnuclei. Insular cortex. The insular cortex can be divided into three subdivisions based on its cytoarchitecture, connections, and physiological responses (Cechetto and Saper, '87). Briefly, the agranular insular cortex is located adjacent to the rhinal sulcus and is distinguished by its lack of a granular layer IV. Immediately dorsal to the agranular region is the dysgranular insular cortex, which contains a poorly differentiated layer IV. The granular insular cortex, most dorsal, possesses a well-defined granular layer.

1 A

B

C

Fig. 5. From rostral to caudal (A-C) camera lucida drawings illustrating the distribution of retrogradely labeled neurons in the subnuclei of the paraventricular hypothalamus after a WGA-HRP injection (R407) into the parabrachial nucleus. Each dot represents one labeled neuron.

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Fig. 6. Polarization photomicrographs of retrograde and axonal labeling in the tuberal lateral hypothalamus (A), and the caudal lateral hypothalamus (B) after a WGA-HRP injection (R407) into the parabrachial nucleus. Bar = 200 pm. T h e high-power, brightfield photo-

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micrographs (C, D) illustrate retrogradely labeled cells present in, respectively, A and B. For orientation, the arrows in A and C, and B and D, indicate the same neuron. Bar = 100 pm.

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639 Rostrally in the insular cortex, the agranular and dysgranular subdivisions are extensive, whereas caudally the granular subdivision predominates, and the agranular/dysgranular subdivisions constitute only a narrow strip. Considering this anatomical heterogeneity, it was not surprising to find that the pattern of anterograde labeling within the P B varied with the insular cortex injection site. Twelve WGA-HRP injections into the insular cortex were studied here. Nine injections were located in rostral levels to midlevels, and the remaining three in the caudal third of the insular cortex. In experiment R87, the injection was centered in the rostral third of the insular cortex and involved primarily the agranular and dysgranular subdivisions (Fig. 8A). In this case, fiber labeling in the rostral P B was particularly dense in the ventral lateral subnucleus, with only scattered fibers in the other subnuclei (Figs. 8B, 9A). At midlevels of the PB, fibers were numerous within the ventral lateral subnucleus and within the portion of the central lateral subnucleus surrounding the internal lateral cell group; a moderate number of fibers was also present in the medial subnucleus (Fig. 8C,D). Most caudally, the waist area contained a small amount of axonal labeling (Fig. 8E). A nearly identical pattern of labeling was noted after an injection, case R32, into the dorsal, granular subdivision of the caudal insular cortex. In this case, labeled fibers were largely confined to the ventral lateral and medial subnuclei although in lesser numbers than after rostral insular cortex injections. In contrast, injections into the ventral caudal half of the insular cortex, here represented by case R31 (Fig. 8F), produced more extensive labeling in the PB. At rostral levels, labeled fibers were found primarily in the ventral lateral and central lateral subnuclei with only scattered fibers elsewhere (Fig. 8G). At midlevels, fiber labeling was widespread with slight concentrations in the ventral lateral and central lateral (surrounding the internal lateral) suhnuclei and in the area between the external lateral and Kolliker-Fuse subnuclei (Figs. 8H, 9B). Axonal labeling was most heavy in the caudal third of the PB, particularly in the medial subnucleus and the waist area (Fig. 81,J).Injections into midrostrocaudal levels of the insular cortex produced a mixed pattern of fiber labeling in the PB. For example, in case R34, there was a heavy concentration of fibers in both the rostral portion of the ventral lateral subnucleus and in the caudal waist area. However, in this case, the slight concentration of fibers seen between the external lateral and Kolliker-Fuse subnuclei after caudal insular cortex injections was absent. Three small PHA-L injections were placed in the insular cortex. All were restricted to its ventral caudal portion. In these cases, fibers with boutons were seen throughout the P B but were concentrated in the ventral lateral and medial subnuclei and in the zone between the external lateral and Kolliker-Fuse subnuclei; this pattern of labeling was identical to that seen in WGA-HRP case R31. In summary, two patterns of labeling in the P B were seen after injections into the insular cortex. Rostra1 and dorsal injections produce heavy fiber labeling in the ventral lateral and central lateral subnuclei; this labeling has the appear-

Fig. 7. Camera lucida drawings illustrating the distribution of retrograde (large dots) and axonal (small dots) labeling in the PB (rostral to caudal, B-E) after a WGA-HRP injection (R218) into the infralimbic cortex. Shaded area in A represents injection site.

H

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Fig. 8. Camera lucida drawings illustrating the distributions of retrograde (large dots) and axonal (small dots) labeling in the PB (rostra1 to caudal, B-E and G-J) after WGA-HRP injections (shaded areas) into A, the rostra1 insular cortex, and F, the caudal insular cortex.

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Fig. 9. Polarization photomicrographs of axonal labeling in the rostral vl subnucleus of the P B (A) after a WGA-HRP injection (R87) into the rostral insular cortex and in the caudal el, med and exm

subnuclei (B) after a WGA-HRP injection (R31) into the caudal insular cortex. In B, note the dense cluster of labeled fibers between the el and KF subnuclei. Bar = 100 fim.

ance of encapsulating the internal lateral subnucleus. In contrast, ventral, caudal injections show more widespread labeling throughout the PB with a particularly heavy concentration of fibers in the caudal waist area and the medial subnucleus and a slight concentration in the area between the external lateral and Kolliker-Fuse subnuclei. Central nucleus of the amygdala/substantia innominata. Ten WGA-HRP and 20 PHA-L injections were placed in either the central nucleus of the amygdala and/or the substantia innominata. Injections into the medial subdivision of the central nucleus of the amygdala, as represented by WGA-HRP case R556 or PHA-L case CP32, produced widespread labeling in the PB. In case R556, the injection site was centered in the rostral part of the medial subdivision with some spread into the medial nucleus of the amygdala (Fig. 10A). Rostrally, labeled fibers were concentrated in the ventral lateral and central lateral subnuclei in a pattern similar to that produced by rostral insular cortex injections (compare Fig. 10B-D with Fig. 8B,C,D). In contrast to the insular cortex cases, the injection in R556 also produced moderately heavy labeling in the external lateral (inner zone) and medial subnuclei, and in the waist area (Fig. 10C-E). In addition, a small number of retrogradely labeled cells was observed in the superior lateral subnucleus (Fig. 10B). A WGA-HRP injection restricted to the medial nucleus of the amygdala (R531) also retrogradely labeled cells in the superior lateral subnucleus, although in larger numbers, suggesting that this subnucleus projects to the medial nucleus rather than to the central nucleus of the amygdala. A PHA-L injection restricted to the medial subdivision of the central nucleus of the amygdala (case CP32) produced identical labeling to that seen after WGA-HRP case R556, further indicating that there was little involvement of the substantia innominata in R556. In this case, fibers with terminal boutons were concentrated in the external lateral (inner zone), ventral lateral, central lateral, and medial subnuclei (Fig. 11)and the waist area. The outer zone of the external lateral subnucleus showed relatively little anterograde labeling as compared to the inner zone. Labeled fibers

in the medial subnucleus had few boutons and resembled fibers of passage. In contrast, injections restricted to the lateral subdivision of the central nucleus of the amygdala (e.g., WGA-HRP case R159 or PHA-L case CP10) produced a very discrete pattern of labeling in the PB. In case R159, a heavy concentration of labeled cells and fibers was present in the external lateral (particularly its outer zone) and waist subnuclei (Fig. 10FJ). Only a few fibers were noted elsewhere in the PB. Likewise, in PHA-L experiment CP10, labeled fibers with boutons were numerous within the external lateral subnucleus and waist area but were scarce throughout the rest of the PB. Substantia innominata injections, as represented by WGAHRP case R135, produced moderately heavy axonal labeling in the external lateral (inner zone) and medial subnuclei and in the waist area, in a pattern similar to that seen after injections into the medial subdivision of the central nucleus (compare Fig. 12C-E with Fig. 10D,E). However, unlike the central nucleus, the substantia innominata sends only light projections to the ventral lateral and central lateral subnuclei (Fig. 12B-D). Furthermore, the substantia innominata receives a heavy input from the PB (Fig. 12B-E; previously described in Fulwiler and Saper, '84) that appears greater than that to either the central nucleus or the bed nucleus of the stria terminalis. Small WGA-HRP injections (3-6 nl) into the PB support the topographical organization reported here. After injections into either the medial subnucleus (R664) or the internal lateralhentral lateral and surrounding central lateral subnuclei (R690), retrogradely labeled cells were numerous throughout the substantia innominata and the medial subdivision of the central nucleus but were largely absent in the lateral subdivision of the central nucleus (Fig. 11B). An injection into the rostral part of the external lateral subnucleus (R622) produced heavy cell and fiber labeling in the lateral subdivision, with only light labeling in the medial subdivision of the central nucleus and in the substantia innominata (Fig. 11C). After a waist area injection (R665), retrograde and axonal labeling was dense throughout both

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Fie. 10. Camera lucida drawinas illustrating the distributions of retrovgrade (large dots) and axon2 (small dot; labeling in the PB (rostra1 to caudal, B-E and G-J) after WGA-HRP injections (shaded

areas) into A, the medial subdivision of the central nucleus of the amygdala (R556),and F,the lateral subdivision of the central nucleus of the amygdala.

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Fig. 11. A Darkfield photomicrograph of anterogradely labeled fibers in a midlevel coronal section of the PB after a PHA-L injection into the medial subdivision of the central nucleus of the amygdala (CP32). Arrows indicate the approximate lateral boundary of the el subnucleus. Note that labeled fibers in the el subnucleus are concen-

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trated medially in i k inner portion. B, C: Polarization photomicrographs of coronal sections through the central nucleus of the amygdala illustrating retrograde and axonal labeling after small WGA-HRP injections into the med subnucleus of the PB (R664) (B), and the el subnucleus of the PB (R622) (C). Bars = 200 pm.

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the substantia innominata and the central nucleus of the amygdala except for a small circular area located in the rostral, lateral part of the central nucleus; this area was previously described as the intermediate subdivision by McDonald (’82). In summary, the lateral subdivision of the central nucleus projects to a limited area in the PB, specifically the outer zone of the external lateral subnucleus and the waist area. The substantia innominata projects to the inner zone of the external lateral subnucleus as well as to the medial and waist subnuclei. The projection from the medial subdivision of the central nucleus to the PB also terminates in the external lateral (inner zone), medial, and waist subnuclei, hut, in addition, it heavily innervates the ventral lateral and central lateral subnuclei. Bed nucleus of the stria terminalis. The bed nucleus of the stria terminalis projection to the PB arises from the dorsal lateral, anterior lateral, posterior lateral, ventral lateral, and preoptic subnuclei (Moga et al., ’89). Twentyfour WGA-HRP and nine PHA-L injections were centered in, or included portions of, the bed nucleus of the stria terminalis. Four different patterns of PB innervation were observed corresponding to injections involving the different BST subnuclei. Two bed nucleus injections (represented by case R527) were centered in the dorsal lateral RST subnucleus with some involvement of the anterior lateral BST subnucleus (Fig. 13A). In these experiments, labeled cells and fibers were concentrated in the external lateral subnucleus and the waist area (Fig. 13C-E). Labeled fibers in the external lateral subnucleus extended medially into the central lateral subnucleus to form a pattern reminiscent of a “wing” (Figs. 13C,D, 14A). This pattern of labeling in the PB was nearly identical to that seen after injections into the lateral subdivision of the central nucleus of the amygdala (compare Fig. IOG-J with Fig. 13B-E). Injections into the posterior lateral subnucleus of the BST, represented by WGA-HRP case R528 (Fig. 13F) and PHA-L case R748, produced a more extensive pattern of axonal labeling in the PB. In case R528, with an injection site centered in the rostral portion of the posterior lateral BST, labeled cells and fibers were concentrated in the external lateral and waist subnuclei in a pattern nearly identical to that found after the dorsal lateral BST injection R527 (Fig. 13H-J); however, unlike R527, axonal labeling was also present in the central lateral, ventral lateral, and medial subnuclei. A PHA-L injection (R748) that was confined to the posterior lateral BST subnucleus produced anterograde labeling in the same PB subnuclei as that in R528. In addition, there was a heavy concentration of fibers with boutons in the ventral lateral subnucleus, which was reminiscent of the pattern of labeling seen after injections into the medial subdivision of the central nucleus of the amygdala (R556), suggesting that case R528 does not fully show the density of the posterior lateral BST projection to the PB. WGA-HRP and PHA-L injections into the ventral subnuclei of the bed nucleus of the stria terminalis produced two

Fig. 12. Camera lucida drawings illustrating the distribution of retrograde (large dots) and axonal (small dots) labeling in the PB (rostral to caudal, B-E) after a WGA-HRP injection (R135) into the substantia innominata. Shaded area in A represents injection site.

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Fig. 14. A, B Polarization photomicrographs of coronal sections through the PB illustrating the dense cell and fiber labeling observed in the el subnucleus after a WGA-HRP injection (R527) into the dorsal lateral subnucleus of the BST (A) and fiber labeling in the rostral cl subnucleus, which largely avoids the underlying vl subnucleus after a WGA-HRP injection (T11) into the preoptic BST (B). In A, note the “wing” of labeled fibers that extends medially into the cl subnucleus. C Darkfield photomicrograph illustrating a dense cluster of labeled terminals in Barrington’s nucleus after a PHA-L injection into the preoptic BST subnucleus. Note also labeled terminals in the central lateral PB subnucleus. Bars = 200 pm.

distinct patterns of labeling in the PB. Rostra1 injections such as R826 (Fig. 15A), which included the ventral lateral BST subnucleus and the parastrial nucleus, showed a relatively discrete pattern of axonal and cellular labeling in the central lateral, external lateral, medial, and waist subnuclei (Fig. 15B,C,E). Retrogradely labeled cells were also present in the extreme lateral, ventral lateral, and dorsal lateral subnuclei; each of these subnuclei contained a few labeled fibers (Fig. 15C,D). After caudal, ventral bed nucleus injections (represented by WGA-HRP case T11 with an injection site centered in the preoptic BST subnucleus and including a small part of the lateral preoptic area), a dense collection of labeled axons was observed in the central gray matter. These labeled axons extended laterally into the PB to form an arc through

the central lateral, superior lateral, and external lateral subnuclei (Figs. 14B, 15G). More caudally, labeled fibers were concentrated in the medial part of the dorsal lateral subnucleus and in the adjacent portion of the central lateral subnucleus (Fig. 15H,I). Small numbers of retrogradely labeled cells were found in the superior lateral, central lateral, dorsal lateral, external lateral, and waist subnuclei (Fig. 15G-J). At the most caudal levels of the PB, labeled fibers in the central gray matter were seen to terminate in a dense cluster coextensive with Barrington’s nucleus, the pontine micturition center (Fig. 151,J; Satoh et al., ’78; Loewy and Saper, ’78). Control injections, located just ventral to the preoptic and ventral lateral BST subnuclei (e.g., case T15) and confined to the lateral preoptic area, also showed an arc of labeled fibers in the rostral PB, although the labeling was less dense than that seen after preoptic BST subnucleus injections. In case T15, unlike T11, there was a “wedge” of dense axonal labeling in the central lateral and dorsal lateral subnuclei between the ventral lateral and external lateral subnuclei and very little labeling in Barrington’s nucleus. In addition, there were numerous retrogradely labeled cells throughout the central lateral and dorsal lateral subnuclei after the lateral preoptic area injection but relatively few after the preoptic BST subnucleus injection. Another indication that the labeling pattern in experiment T11 was specific to the preoptic BST subnucleus came from PHA-L experiment R779. In this case, the injection site was limited to the preoptic BST subnucleus; labeled fibers with boutons were particularly dense in the central lateral subnucleus and in Barrington’s nucleus, in a pattern identical to that seen in T11 (Fig. 14C). Small

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WGA-HRP injections into Barrington’s nucleus (R594, R599) retrogradely labeled a continuum of neurons extending from the anteroventral periventricular nucleus, through the medial preoptic nucleus, to the posterodorsal nucleus and preoptic subnucleus of the BST. In these cases, very few retrogradely labeled cells were located in the lateral preoptic area beneath the BST. Thus the connections of the preoptic BST subnucleus and the lateral preoptic area with the P B can be distinguished from one another, although the differences are relatively subtle. Small WGA-HRP injections into the PB confirmed our findings regarding the terminal fields of the BST subnuclei. After injections into the external lateral PB subnucleus (R622, R683), dense clusters of labeled cells and fibers were observed in the ventral lateral and dorsal lateral BST subnuclei, with only a scattering of labeled cells in the anterior lateral and posterior lateral BST subnuclei. In contrast, injections into either the medial subnucleus (R664) or the internal lateral/ventral lateral and surrounding central lateral subnuclei (R690) produced heavy cell labeling in the posterior lateral and anterior lateral BST subnuclei but only a few labeled cells in the dorsal lateral and ventral lateral BST. After an injection into the waist area (R665), numerous labeled cells and fibers were observed throughout the dorsal lateral, ventral lateral, anterior lateral, and posterior lateral BST subnuclei. Labeled cells were seen in the preoptic BST subnucleus after injections that included the central lateral (R645, R683, R690) or dorsal lateral subnuclei (R614). In summary (Fig. 16A,B), the dorsal lateral BST subnucleus projects primarily to the external lateral and waist subnuclei. The posterior lateral BST subnucleus also projects to these subnuclei but, in addition, sends moderately heavy projections to the medial, central lateral, and ventral lateral subnuclei. The anterior lateral BST subnucleus projects to the same PB subnuclei as the posterior lateral BST subnucleus except for the external lateral PB subnucleus. The

ventral lateral BST subnucleus sends inputs to the external lateral, medial, and waist subnuclei that are similar in distribution to the projections of the dorsal lateral BST and the central nucleus of the amygdala; in addition, it innervates the central lateral, external lateral, and dorsal lateral subnuclei in a pattern similar to that of certain hypothalamic nuclei (see below). Likewise, the preoptic BST subnucleus also projects to the P B in a “hypothalamic manner,” terminating heavily in the central lateral, dorsal lateral, superior lateral, and external lateral subnuclei. Paraventricular hypothalamus. Seven animals received injections of WGA-HRP that included the paraventricular nucleus of the hypothalamus. Two injections were centered in the caudal half of the paraventricular nucleus, one in the rostral half, and four in adjacent nuclei (i.e., anterior hypothalamic area, zona incerta) but with substantial involvement of the paraventricular nucleus. The pattern of axonal labeling in the PB was identical for each case. In case AP3, in which the injection was most closely confined to the paraventricular nucleus, many labeled fibers were found rostrally in the central lateral, external lateral, and medial subnuclei (Fig. 17A,B).Both cells and fibers were present in the superior lateral and extreme lateral subnuclei (Fig. 17B). At mid-PB levels, light cell and fiber labeling was found in the dorsal lateral subnucleus and along the surface of the superior cerebellar peduncle in the external lateral subnucleus (Fig. 17C,D). In the most caudalPB, a concentration of axonal labeling was observed in the waist area between the superior cerebellar peduncle and the mesencephalic nucleus and tract of the trigeminal nerve (Fig. 17E).

Retrochiasmatic area/uentromedial hypothalamus. In the retrograde experiments, labeled cells in the retrochiasmatic area were observed to extend caudally into the ventrolateral portion of the ventromedial nucleus of the hypothalamus, suggesting a continuum of cells projecting to the PB. Of the seven WGA-HRP and two PHA-L injections placed in either the retrochiasmatic area and/or the ventro-

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medial nucleus, all produced an identical pattern of anterograde labeling in the PB; thus we consider the cells in the retrochiasmatic area and ventromedial nucleus that project to the P B to constitute a single population. In case R627, in which the WGA-HRP injection was centered in the retrochiasmatic area (Fig. 17F), there was nearly complete overlap of axonal and cellular labeling; both were found in the dorsal lateral, central lateral, and superior lateral subnuclei (Fig. 17G-I). Of these, the dorsal lateral subnucleus contained the heaviest fiber labeling (Figs. 17H, 18A). The pattern of axonal labeling was more easily seen in the PHA-L experiments (represented by R825), where there was no retrograde labeling. In case R825, with an injection site centered in the lateral part of the retrochiasmatic area, a small cluster of labeled fibers with boutons was observed in the rostral portion of the central lateral subnucleus; these fibers travelled caudally to terminate in the medial portion of the

dorsal lateral subnucleus as well as in the adjacent central lateral subnucleus. Scattered axons with boutons were also noted in the medial, ventral lateral, superior lateral, and central lateral (surrounding the internal lateral) subnuclei. In experiment R629, where the WGA-HRP injection site was centered in the rostral portion of the ventromedial nucleus, a similar pattern of anterograde labeling was found. However, in this case the pattern of retrograde labeling was not as extensive, being confined largely to the superior lateral subnucleus (see Fulwiler and Saper, ’85). In summary, the retrochiasmatic area and ventromedial nucleus send moderately light projections to the central lateral, dorsal lateral, and superior lateral subnuclei. Median preoptic nucleus. Three WGA-HRP injections were centered in the median preoptic nucleus. Axonal labeling in the PB was identical in all three experiments. In representative case R154, light fiber labeling in the central

Fig. 18. Polarization photomicrographs of WGA-HRP labeling in the PB after injections into the hypothalamus. A A dense collection of retrogradely labeled cells, and a few labeled fibers, in the dl subnucleus after an injection into the retrochiasmatic area (R627). B: A cluster of labeled cells and fibers in the medial portion of the dl subnucleus adjacent to the il subnucleus after an injection into the dorsomedial

nucleus of the hypothalamus (R563). C A “wedge” of retrograde and axonal labeling in the cl and dl subnuclei between the vl and el subnuclei after an injection into the tuberal lateral hypothalamus (R93).D: Cell and fiber labeling in the waist area of the caudal PB after an injection into the caudal lateral hypothalamus (R121). Bar = 100 pm.

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PARABRACHIAL FOREBRAIN AFFERENTS lateral subnucleus was noted at rostral levels of the P B (Fig. 19B). At the midlevel of the PB, fibers were found amidst a cluster of labeled cells located in the central lateral subnucleus adjacent to the internal lateral subnucleus; these cells and fibers extended laterally into the rostral third of the external lateral subnucleus (Fig. 19C). Anterograde labeling was also present in the dorsal lateral subnucleus throughout its rostrocaudal extent (Fig. 19D). Three PHA-L injections were placed into the median preoptic nucleus. In all three cases, the pattern of fiber labeling in the PB was identical to that seen after the WGA-HRP injections. In experiment R272, fibers with terminal boutons were particularly numerous in the central lateral, the dorsal lateral, and the rostral part of the external lateral subnuclei. Dorsomedial hypothalamus. In four WGA-HRP experiments, the injections involved the dorsomedial nucleus of the hypothalamus. In each case, the injection site included a portion of the adjacent lateral hypothalamus, although in experiment R563 there was only minimal extension beyond the borders of the dorsomedial nucleus (Fig. 19F). In this case, a moderately dense band of cell and fiber labeling was seen arcing rostrally across the central lateral subnucleus (Fig. 19G). At midlevels of the PB, a cluster of labeled cells and fibers was located in the central lateral and dorsal lateral subnuclei adjacent to the internal lateral subnucleus (Figs. 18B, 19H). More caudally, labeled cells and fibers were concentrated in the central lateral and dorsal lateral subnuclei, in a “wedge” pattern between the ventral lateral and external lateral subnuclei (Fig. 191). The pattern of labeling in R563 was similar to that seen following injections into the adjacent lateral hypothalamus (see below) but was more discrete and largely excluded the medial, superior lateral, and extreme lateral subnuclei, which receive heavy input from the lateral hypothalamus. Furthermore, the dense cluster of labeled cells and fibers seen adjacent to the internal lateral subnucleus in experiment R563 (Fig. 18B) was absent after lateral hypothalamus injections. Thus the projections to the central lateral, dorsal lateral, and rostral external lateral subnuclei seen in R563 probably originate from the dorsomedial nucleus. Lateral hypothalamus. In 19 experiments (14 WGAHRP, five PHA-L), injections were placed into the lateral hypothalamus. After a WGA-HRP injection into the rostral lateral hypothalamus (R408),only a small number of fibers were labeled in the PB, mainly in the central lateral and medial subnuclei. In contrast, injections into the tuberal lateral hypothalamus, represented by experiment R93 (Fig. 20A), produced a heavy arc of cellular and fiber labeling in the rostral PB extending through the central lateral, superior lateral, and external lateral subnuclei (Fig. 20B). At mid-PB levels, labeled cells and fibers were concentrated in the central lateral and dorsal lateral subnuclei in a “wedge” pattern between the external lateral and ventral lateral subnuclei (Figs. 18C, 20C,D). Labeling was sparse elsewhere in the PB, particularly in the caudal subnuclei (Fig. 20E). Caudal lateral hypothalamus injections, represented by experiment R121 (Fig. ZOF), produced a slightly different pattern of fiber labeling in the PB. In this case, there appeared to be less anterograde labeling in the rostral PB subnuclei (e.g., central lateral, superior lateral), and more labeling in the external lateral subnucleus and the waist area, than that seen after tuberal lateral hypothalamus injections (Figs. 18D,20G-J). Two caudal lateral hypothala-

mus injections, C30 and R437, included the overlying zona incerta. In these cases, additional fiber labeling was seen in the rostral portion of the ventral lateral subnucleus. Small WGA-HRP injections into the YB confirmed these findings. After an injection restricted to the waist area (R665), WGA-HRP-labeled cells and fibers were found solely in the caudal part of the lateral hypothalamus adjacent to the subthalamic nucleus. Injections into the external lateral subnucleus (R622) and the internal lateralhentral lateral and surrounding central lateral subnuclei (R690) produced labeled cells in both the tuberal and the caudal lateral hypothalamus. In summary, the PB receives input from both the tuberal and the caudal lateral hypothalamus. The tuberal lateral hypothalamus projects heavily to the rostral PB; its fibers form a heavy arc of labeling through the lateral P B subnuclei. In contrast, the caudal lateral hypothalamus sends moderately dense projections to both the rostral PB subnuclei and the waist area.

DISCUSSION Our results show that the forebrain afferents to the PB terminate in three relatively discrete patterns, corresponding to the three major sources of forebrain input to the PB, namely, the basal forebrain, hypothalamus, and cerebral cortex (Fig. 21). The basal forebrain projection to the PB arises from a continuum of neurons spanning the bed nucleus of the stria terminalis, the substantia innominata, and the central nucleus of the amygdala. This projection heavily innervates the external lateral and waist subnuclei. The hypothalamic innervation of the PB arises from cell populations in the median preoptic, medial preoptic, paraventricular, dorsomedial, and ventromedial nuclei and in the lateral preoptic, retrochiasmatic, lateral, and dorsal hypothalamic areas. Hypothalamic afferents innervate predominantly rostral portions of the PB, particularly the central lateral and dorsal lateral subnuclei. The cortical projection to the PB originates from neurons in the infralimbic, lateral prefrontal, and insular cortices. Of the three forebrain areas projecting to the PB, the cortical afferents showed the most diffuse labeling in the PB; however, concentrations of labeled fibers were found rostrally in the ventral lateral and central lateral subnuclei, and caudally in the medial subnucleus. In general, each region was found to innervate the same PB subnuclei from which it receives its input.

Basal forebrain projections to the PB A continuum of retrogradely labeled cells in the central nucleus of the amygdala, substantia innominata, and bed nucleus of the stria terminalis was previously reported after injections into the parabrachial area (Jackson and Crossman, ’81; de Olmos et al., ’85). Anatomists have long emphasized the continuity of these three cell groups based on their similar cytoarchitecture, immunocytochemistry and connections (Johnston, ’23; Schwaber et al., ’82; Holstege et al., ’85;de Olmos et al., ’85; Moga et al., ’89; Shimada et al., ’89). Our findings in the present study support the close anatomical ties described for these nuclei. We observed three patterns of axonal labeling in the PB after injections into the basal forebrain; two of these patterns (as represented by R556, R135, and R528 and by R159 and R527) were observed after injections into the central nu-

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PARABRACHIAL FOREBRAIN AFFERENTS cleus of the amygdala, the substantia innominata, and the dorsal part of the bed nucleus of stria terminalis, and the third pattern (represented by R826 and T11) was seen only after injections into the ventral portion of the bed nucleus of the stria terminalis, an area that may be more closely associated with the adjoining preoptic area. The central nucleus of t h e amygdala has long been recognized as the major amygdaloid source of input to the P B in the rat, cat and monkey (Krettek and Price, ’78; Hopkins and Holstege, ’78; Hopkins e t al., ’81; Price and Amaral, ’81; Price, ’81; Veening e t al., ’84). In these early studies, no topographic organization was reported within this pathway. Krettek and Price (’78) observed that fibers from the central nucleus in the rat were “diffusely distributed around cells both dorsal and ventral to the brachium conjunctivum.” Likewise, Price and Amaral (’81)found this projection in the monkey to “ramify extensively in both medial and lateral PB.” Following injections of a retrograde fluorescent tracer into the PB, Veening et al. (’84) observed retrogradely labeled neurons concentrated within the medial subdivision of the central nucleus, with few labeled neurons in the lateral subdivision. This pattern of retrograde labeling was nearly identical to that reported in the central nucleus after injections into the nucleus of the solitary tract, suggesting that the medial subdivision was the major amygdaloid source of afferents to autonomic nuclei in the brainstem (Schwaber et al., ’82). We noted a similar cell distribution in the medial subdivision of the central nucleus after retrograde tracer injections placed medially in the PB; however, we also observed numerous retrogradely labeled cells in the lateral subdivision following injections into the ventrolatera1 part of the P B (i.e., the external lateral subnucleus), suggesting topographic differences between the projections of the medial and lateral subdivisions of the central nucleus (Moga and Gray, ’85b). We were able to confirm and extend these findings in the present study; specifically, we observed that the lateral subdivision of the central nucleus projects to a restricted area within the PB (the outer zone of the external lateral subnucleus and the waist area), whereas the medial subdivision of the central nucleus projects more widely within the P B (the external lateral (inner zone), central lateral, ventral lateral, medial, and waist subnuclei. I t is likely that the PB injection sites of Veening e t al. (’84), which labeled very few neurons in the lateral subdivision of the central nucleus, did not include the external lateral and waist subnuclei. Neurons in the central nucleus of the amygdala stain with antisera against a wide variety of neuropeptides; many of these neurons have been found to project to the PB. Approximately 30-60% of the corticotropin-releasing factor, neurotensin-, and somatostatin-like immunoreactive neurons in the central nucleus innervate the P B (Moga and Gray, ’85b; Milner and Pickel: ’86a; Sakanaka et al., ’86). A few substance P- and galanin-like immunoreactive neurons in the medial subdivision also contribute to this pathway (Milner and Pickel, ’86b; Gray and Magnuson, ’87a). The

Fig. 19. Camera lucida drawings illustrating th e distributions of retrograde (large dots) and axonal (small dots) labeling in the PB (rostra1 to caudal, B-E and G-J) after WGA-HRP injections (shaded areas) into A, the median preoptic nucleus of the hypothalamus (R154), and F, the dorsomedial nucleus of the hypothalamus (R563).

653 distributions of corticotropin releasing factor- and neurotensin-like immunoreactive neurons and of substance P- and somatostatin-like immunoreactive neurons in the central nucleus strongly overlap, suggesting some degree of neuropeptide coexistence. Indeed, Shimada et al. (’89) recently reported that a majority of the corticotropin-releasing factorlike immunoreactive neurons in the lateral subdivision stain with antisera against neurotensin and that most of the substance P-like immunoreactive neurons in the medial subdivision also stain with antisera against somatostatin. Some of these double-labeled neurons probably project to the PB; however, this possibility has not been examined. Neurons in the intercalated nuclei of t h e amygdala also project to the PB, and some of these neurons are immunoreactive with antisera against neurotensin, somatostatin, or corticotropin-releasing factor (Moga and Gray, ’85a). In the present study, we observed that this projection arises from a t least four different intercalated cell masses. All the retrogradely labeled intercalated neurons were smaller than labeled neurons in the central nucleus; these neurons may correspond to the medium intercalated neurons described by Millhouse (’86). Surprisingly, individual WGA-HRP experiments with similar injection sites in the PB often labeled different intercalated cell groups, suggesting that the P B terminal fields of these cell groups are quite small and discrete. The P B receives input from the anterodorsal part of the sublenticular substantia innominata (de Olmos et al., ‘85; Grove, ’88; present study). Grove (’88) observed that “the subcortical projections of the dorsal substantia innominata are strikingly reminiscent of those reported for the lateral bed nucleus of the stria terminalis and central nucleus of the amygdala.” We found that the P B terminal field of the dorsal substantia innominata is similar to that of the medial subdivision of the central nucleus and the posterior lateral subnucleus of the bed nucleus of the stria terminalis in its innervation of the inner zone of the external lateral subnucleus and of the waist area. However, unlike these areas, the substantia innominata does not diffusely innervate the central lateral, ventral lateral, and medial subnuclei. Although previous studies demonstrated projections from the bed nucleus of t h e stria terminalis to the PB, they did not describe in detail the PB terminal distribution (Swanson and Cowan, ’79; van der Kooy et al., ’84; Holstege et al., ’85). The BST projection to the P B arises from cells in the dorsal lateral, anterior lateral, posterior lateral, ventral lateral, and preoptic subnuclei (Moga et al., ’89). After injections into the dorsal lateral BST subnucleus, dense clusters of labeled fibers were present in the external lateral and waist subnuclei in a pattern identical to that seen after injections into the lateral subdivision of the central nucleus of the amygdala. Furthermore, many PB-projecting cells in both the dorsal lateral BST and the lateral subdivision of the central nucleus are immunoreactive to corticotropin-releasing factor, neurotensin, or somatostatin antisera (Moga and Gray, ’85b; Moga et al., ’89). Like the lateral subdivision of the central nucleus, most of the corticotropinreleasing factor-like immunoreactive neurons in the dorsal lateral BST stain for neurotensin-like immunoreactivity (Shimada et al., ’89). Furthermore, cells in these two subdivisions are quite similar in size, shape, and Nisslstaining characteristics when the BST is viewed in the coronal plane and the central nucleus in the sagittal plane,

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E Fig. 20. Camera lucida drawings illustrating the distributions of retrograde (large dots) and axonal (small dots) labeling in the PB (rostra1 to caudal, B-E and G-J) after WGA-HRP injections (shaded areas) into A, the tuberal lateral hypothalamus (R93j, and F,the caudal lateral hypothalamus (R121j.

PARABRACHIAL FOREBRAIN AFFERENTS central lateral

V l

internal lateral

Kolliker-Fuse ventral lateral

u

665

suggesting a splitting and 90° shift of these nuclei during development (Moga, unpublished observations). The terminal field of the posterior lateral BST included the external lateral and waist subnuclei but, in addition, extended medially into the central lateral, ventral lateral, and medial subnuclei in a manner very similar to that of the medial subdivision of the central nucleus of the amygdala. In addition to their relatively diffuse projections to the PB, both the posterior lateral BST and the medial subdivision of the central nucleus also project to the nucleus of the solitary tract and to the ventral lateral medulla (Gray and Magnuson, ’87b; Moga et al., ’89). Like the medial part of the central nucleus, many of the substance P-like immunoreactive neurons in the posterior lateral BST stain for somatostatin-like immunoreactivity (Shimada et al., ’89). These results support the homology previously noted between these two structures (Holstege et al., ’85; Gray and Magnuson, ’87b;Moga et al., ’89). The ventral lateral BST subnucleus and the adjacent parastrial nucleus project to the P B in a pattern that has both hypothalamic and amygdaloid characteristics. These two cell groups innervate the external lateral subnucleus, the adjoining “wing” of the central lateral subnucleus, and the waist area in a manner similar to that of the lateral subdivision of the central nucleus and the dorsal lateral BST and the central lateral, external lateral, medial, and dorsal lateral subnuclei in a pattern similar to that observed after injections into the median preoptic and dorsomedial nuclei of the hypothalamus (compare case R826 in Fig. 14 with R159 and R528 in Figs. 9 and 12 and with R154 and R563 in Fig. 17). Unlike the other lateral BST subnuclei, both the ventral lateral BST and the parastrial nucleus are reciprocally connected with the paraventricular and dorsomedial nuclei of the hypothalamus (Simerly and Swanson, ’88; Moga, unpublished observations). As the ventral lateral BST receives a particularly heavy input from the medial portion of the nucleus of the solitary tract (Zardetto-Smith and Gray, ’87), this area of the BST may be important in conveying general visceral information to autonomic nuclei in the hypothalamus. Axonal labeling in the PB after preoptic BST injections was concentrated rostrally in an “arc” spanning the central lateral, superior lateral, and external lateral subnuclei. This pattern resembles that seen after tuberal lateral hypothalamic injections (for comparison, see T11 and R93 in Figs. 14 and 18). This area of the BST, straddling the boundary between diencephalon and telencephalon, was described as the preoptic continuum of the BST by Swanson (’76) based on its input from the stria terminalis and on its cytoarchitecture, which resembles that of the underlying preoptic neurons. Our WGA-HRP and PHA-L experiments suggest that this cell group may more properly belong with the preoptic hypothalamus than with the BST.

Fig. 21. Summary diagram depicting the major terminal fields of the hypothalamic (dots), cortical (hatching) and amygdaloid (stippling) afferents to the parabrachial nucleus (rostra1 to caudal, top to bottom). Note that the different forebrain sites mark out largely nonoverlapping terminal fields that taken together subtend nearly all of the PB complex and that the internal lateral and Kolliker-Fuse subnuclei (unshaded) receive little afferent input from the forebrain.

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Hypothalamic projection to the PB Hypothalamic projections to the PB were previously shown to arise from the medial preoptic, median preoptic, paraventricular, dorsomedial, and ventromedial nuclei and from the lateral hypothalamic area (Conrad and Pfaff, ’76; Swanson, ’77; Saper et al., ’76, ’79; Hosoya and Matsushita, ’81; Berk and Finkelstein, ’82; Saper and Levisohn, ’83; Takeuchi and Hopkins, ’84; Chiba and Murata, ’85; Luiten et al., ’85; ter Horst and Luiten, ’86; Veening et al., ‘87; Simerly and Swanson, ’88). In the present study, we found that cells in the retrochiasmatic area, dorsal and posterior hypothalamic areas, and lateral preoptic area also contribute to the hypothalamic-PB pathway. Our results indicate that each hypothalamic afferent has a unique pattern of innervation within the PB, although the terminal zones of these afferents show considerable overlap. The projection from the lateral hypothalamus to the PB is particularly dense and originates from two cell populations, one in the tuberal lateral hypothalamus, dorsal and lateral to the fornix, and the other in the caudal lateral hypothalamus, adjacent to the subthalamic nucleus and the internal capsule; these two cell populations innervate different, yet overlapping, subsets of PB subnuclei (present study; Veening et al., ’87). Saper et al. (’79) previously noted topographical differences between the projections from the tuberal and caudal lateral hypothalamic areas. They observed axonal labeling in the PB after caudal but not after tuberal injections, possibly because their tuberal lateral hypothalamus injections were centered ventral to the fornix, an area with little input to the PB. In the present study, injections into the dorsolateral tuberal area of the lateral hypothalamus produced a heavy “arc” of fiber labeling in the rostral PB, spanning the central lateral, superior lateral, extreme lateral, and external lateral subnuclei. Earlier studies noted a similar pattern of fiber labeling coursing “posteriorly through the central tegmental tract, ventral to the cuneiform nucleus, and travel[ing] into the lateral and medial PB” (Berk and Finkelstein, ’82; for a similar description, see Hosoya and Matsushita, ’81). These fibers terminate at mid-PB levels in the central lateral and dorsal lateral subnuclei, largely avoiding the adjacent ventral lateral and external lateral subnuclei, thus producing a “wedge” pattern of cellular and fiber labeling (Fig. 20C). The caudal lateral hypothalamus projects less heavily to the same PB subnuclei as the tuberal lateral hypothalamus; in addition, it innervates the inner zone of the external lateral subnucleus and the waist area in a pattern similar to that seen after injections into the medial subdivision of the central nucleus of the amygdala. Interestingly, the caudal lateral hypothalamus and the medial subdivision of the central nucleus of the amygdala, besides being reciprocally connected with the external lateral and waist subnuclei, are also reciprocally connected with each other (Krettek and Price, ’78; Saper et al., ’79). Recently, we found that these two lateral hypothalamic pathways to the PB have different neurochemical compositions (Moga et al., ’90). Specifically, many dynorphin-, neurotensin-, substance P-, corticotropin-releasing factor-, and angiotensin 11-immunoreactiveneurons in the perifornical region of the tuberal lateral hypothalamus project to the PB. In the tuberal area adjacent to the internal capsule, many a-melanocyte-stimulating hormone-like immunoreactive neurons are also retrogradely labeled following P B

injections; these double-labeled cells extend for a short distance into the caudal lateral hypothalamus. In general, there is very little immunoreactive staining for any known neuropeptide in the caudal lateral hypothalamus, so the neurotransmitter content of its pathways is essentially unknown. Few details have been reported concerning the projection from the parauentricular nucleus of the hypothalamus to the PB (Saper et al., ’76; Takeuchi and Hopkins, ’84; Luiten et. al., ’85). Following lesions of the paraventricular nucleus and the immediately adjacent lateral hypothalamus, Takeuchi and Hopkins (’84) observed numerous degenerating axons in the rostral half of the lateral PB and a less dense, more diffuse distribution of degenerating terminals in the medial PB, a pattern of labeling similar to our present findings. However, we observed two additional areas of fiber concentration within the PB, one in the rostral part of the external lateral subnucleus and the other in the waist area between the superior cerebellar peduncle and the mesencephalic tract and nucleus of the trigeminal nerve. The axonal labeling in the external lateral subnucleus was continuous with labeling in the adjacent pedunculopontine nucleus and central tegmental tract. This labeling may correspond to the “strongly branching and terminating” projection to the region of the pedunculopontine nucleus observed by Luiten et al. (’85)following PHA-L injections into the paraventricular nucleus. Interestingly, neurophysin-immunoreactive fibers are concentrated in the waist area of the PB in a pattern identical to that of the axonal labeling seen after injections into the paraventricular hypothalamus (Swanson, ’77), suggesting the presence of this peptide in the pathway from the paraventricular hypothalamus to the PB. Indeed, we have recently found retrogradely labeled vasopressinand oxytocin-like immunoreactive neurons in the paraventricular nucleus after tracer injections into the PB (Moga et al., ’90). In agreement with findings of Takeuchi and Hopkins (’84),the PB receives a small input from neurons in the ventromedial hypothalamic nucleus. In an earlier study of the ventromedial hypothalamic efferents, Saper et al. (’76) did not detect any fibers in the PB; this was probably due to the very small number of neurons contributing to this pathway and to the relatively less sensitive autoradiographic techniques used in those early experiments. Neurons in the rostral, ventrolateral part of the ventromedial nucleus that project to the PB are identical in size and appearance to PB projecting neurons in the adjacent retrochiasmatic area, suggesting that these neurons belong to a single population. More conclusively, both the retrochiasmatic area and the ventromedial nucleus project to identical terminal fields in the PB, namely, the central lateral, superior lateral, and dorsal lateral subnuclei. A similarly distributed population of retrochiasmatic and ventromedial neurons has previously been shown to project to other autonomic nuclei, such as the dorsal vagal complex and the spinal cord (Hosoya, ’80; Swanson and Kuypers, ’80; Cechetto and Saper, ’88). Like the ventromedial nucleus, the dorsomedial nucleus of the hypothalamus also sends a light projection to the PB (Takeuchi and Hopkins, ’84; ter Horst and Luiten, ’86). After P B injections, retrogradely labeled neurons in the large-celled portion of the dorsomedial nucleus appeared similar to, and continuous with, labeled cells in the adjacent lateral hypothalamus, suggesting an anatomical continuum

PARABRACHIAL FOREBRAIN AFFERENTS between these nuclei; however, in this case the PB terminal fields of these two nuclei were distinctly different. The lateral hypothalamus projection was diffuse and extensive and produced a heavy “arc” of fiber labeling in the rostral PB. In contrast, the dorsomedial nucleus projection was discrete and labeled a tight cluster of fibers in the rostral part of the central lateral subnucleus. This cluster terminated at mid-PB levels in a dense “spot” of labeling in the medial part of the dorsal lateral subnucleus and in the adjacent portion of the central lateral subnucleus. The dorsomedial nucleus terminal field was nearly identical to that seen after injections into the median preoptic nucleus, with the exception of additional labeling seen in the central lateral subnucleus at mid-PB levels after dorsomedial nucleus injections, suggesting some possible functional similarities in these pathways. The PB receives input from nuclei throughout the preoptic region; however, the major sources of this input are the median preoptic nucleus, the medial preoptic nucleus, and the lateral preoptic area. The reciprocal connections of the median preoptic nucleus and PB were described in earlier studies (Saper and Levisohn, ’83; Fulwiler and Saper, ’84). The results of the present study further emphasize the close association of the anterograde and retrograde labeling in the P B after median preoptic injections and the discreteness of the median preoptic terminal field in rostral parts of the central lateral and external lateral subnuclei and in the dorsal lateral subnucleus. In an early study of the efferents from the medial preoptic area, Conrad and Pfaff (’76) noted that “at the rostral border of the pons, some labeled fibers in the central grey shifted laterally, into the parabrachial nucleus and nucleus cuneiformis.” This labeling was not reported by Swanson (’76), whose injection sites in the medial preoptic area were located more rostrally and medially, involving primarily the anteroventral periventricular and suprachiasmatic nuclei. Our retrograde transport experiments show labeled neurons in the lateral part of the medial preoptic area (Fig. 1E,F), primarily along the lateral border of the medial preoptic nucleus. In support of these findings, Simerly and Swanson (’88), in their PHA-L study of medial preoptic efferents, found that labeled terminals in the PB were most numerous after injections into the lateral subdivision of the medial preoptic nucleus. Furthermore, they demonstrated labeled terminals in the central lateral and dorsal lateral subnuclei of the PB in the “wedge” pattern characteristic of other hypothalamic afferents. Interestingly, all three anterograde studies (Conrad and Pfaff, ’76; Swanson, ’76; Simerly and Swanson, ’88) noted descending fibers from the medial preoptic area terminating in a region just medial and anterior to the locus coeruleus and medial to the mesencephalic nucleus and tract of the trigeminal nerve. This area may correspond to Barrington’s nucleus. After small WGA-HRP injections into Barrington’s nucleus, we have observed a large number of retrogradely labeled cells throughout the medial preoptic area. Scattered neurons in the lateral preoptic area, amidst fibers of the medial forebrain bundle, also provide an input to the PB. After injections into the lateral preoptic area, an “arc” of cell and fiber labeling that spanned the central lateral, superior lateral, external lateral, and extreme lateral subnuclei was observed in the rostral PB. A t mid-PB levels, cell and fiber labeling was concentrated in the central lateral and dorsal lateral subnuclei, in a wedge-like pattern reminis-

657 cent of the tuberal lateral hypothalamus terminal field. In his study of preoptic efferents, Swanson (”76) did not describe this projection, possibly because of its diffuse origin and the small size of his injection sites or because of the lower sensitivity of the autoradiographic technique used. Although there is substantial overlap of the PB terminal fields originating in the hypothalamus, there appears to be considerable neurochemical specificity within each hypothalamic projection. For example, a majority of cells contributing to this projection from both the arcuate nucleus and the retrochiasmatic area are immunoreactive with antisera against proopiomelanocortin-derived peptides (Moga et al., ’90).However, many dynorphin-, angiotensin-, and neurotensin-like immunoreactive neurons in the retrochiasmatic area, but not in the arcuate nucleus, also project to the PB. The tuberal lateral hypothalamus projection to the PB arises from two different neuropeptide-immunoreactive cell populations (as noted above), one along the internal capsule that is largely a-melanocyte-stimulating hormone-like immunoreactive and the other around the fornix that contains dynorphin-, galanin-, neurotensin-, angiotensin-, and corticotropin-releasing factor-like immunoreactive neurons. Small numbers of neurons in the paraventricular hypothalamus that project to the P B react with each of a wide variety of neuropeptide antisera; of these, the met-enkephalin- and dynorphin-like immunoreactive neurons are the most numerous.

Cortical projection to the PB In general, cortical afferents innervate the PB in a more diffuse manner than either hypothalamic or amygdaloid inputs. However, each of the three cortical areas projecting to the PB (the infralimbic, rostral insularhatera1 frontal, and caudal insular areas) shows a unique terminal distribution within the PB. Early studies on the relationship between the PB and the insular cortex emphasized that the insular cortical afferents to the PB are distributed in a similar pattern to that of the cortical efferents (Saper, ’82; Shipley and Sanders, ’82). In addition, Saper (’82) further noted that injections of WGAHRP into the dorsal part of the insular cortex produce axonal labeling primarily in the ventral and medial parts of the PB, whereas more ventral insular injections produce more widespread labeling, including much of the lateral PB. The current study confirms and extends these observations. After injections into the ventral, caudal insular cortex, we observed widespread labeling throughout the PB, with a particularly heavy concentration of fibers in the medial and caudal PB subnuclei, areas with a high density of retrogradely labeled cells. We also found a more restricted pattern of labeling after both rostral insular and dorsal, caudal insular injections. Specifically, in these cases labeled fibers were limited to the medial and ventral lateral subnuclei and to that portion of the central lateral subnucleus that surrounds the internal lateral subnucleus. These ventraldorsal and rostral-caudal topographic differences may reflect the functional heterogeneity of the different parts of the insular cortex (Cechetto and Saper, ’87; Yasui et al., ’90). For example, taste- and gastric mechanoreceptor-responsive neurons are concentrated in the rostral and dorsal, caudal areas of the insular cortex, whereas baroreceptorand chemoreceptor-responsive neurons are most common in the caudal, ventral portion of the insular cortex (Cechetto and Saper, ’87).

658 Previous reports on the projection from the infralimbic cortpr to the PB have not described its terminal distribution (Saper, '82; Terreberry and Neafsey, '87;van der Kooy et al., '84). Our results show that this projection terminates diffusely within the PB, with little organization. We did, however, see a cluster of axonal labeling in the most medial portion of the medial subnucleus; this labeling may correspond to the concentration of fibers reported in the medial PB by van der Kooy et al. ('84). Our PHA-L experiments suggest that most of the fibers in this area are fibers of passage. In general, the infralimbic terminal field is most easily characterized by the subnuclei it excludes, namely, the internal lateral and to a lesser extent the ventral lateral and superior lateral subnuclei. This was most apparent after large WGA-HRP injections. After WGA-HRP injections into the infralimbic cortex, orbital gyrus and lateral bank of the presylvian gyrus of the cat, Yasui et al. ('85) observed three patterns of axonal labeling in the PB that were similar to those seen in the present study, suggesting homologous PB subnuclei in the cat. Like the rat, the projection in the cat from the infralimbic cortex to the PB is diffuse and moderately heavy. Injections into the lateral bank of the presylvian gyrus, which receives gustatory input from the thalamus, produce a discrete pattern of axonal labeling in the PB that resembles the rostral ventral lateral/central lateral subnuclear labeling observed after rostral and dorsal insular cortex injections in the rat. Orbital cortex injections also label fibers terminating in this area of the P B but to a lesser extent. In addition, they label numerous fibers in the medial PB and waist area; this labeling is similar to that seen after caudal insular injections in the rat. Using the combined retrograde fluorescence-immunofluorescence method, we have not found any neuropeptideimmunoreactive cells in the cortex that project to the PB (Moga,unpublished observations). Most of the neuropeptideimmunoreactive neurons in the cortex are y-aminobutyric acid (GABA)ergicor resemble local circuit neurons (Hendry et al., '84; Schmechel et al., '84), so the lack of a descending, peptidergic projection to the PB is not surprising.

Reciprocal forebrainconnections of the PB Unlike the medullary connections, all the forebrain connections of the P B are reciprocated, with the exception of the superior lateral projection to the medial nucleus of the amygdala (Herbert et al., '90). The degree of reciprocity, that is, the degree of association between the efferent neurons and the afferent fibers, varied for each P B forebrain connection. Some PB afferent fibers, such as those from the median preoptic nucleus, retrochiasmatic area, and lateral subdivision of the central nucleus, were very tightly associated with their respective PB efferent neurons. This reciprocity has been confirmed at the cellular level in an electron microscopic study that showed axons from the central nucleus of the amygdala synapsing onto PB neurons that project to the central nucleus (Takeuchi et al, '82). In contrast, the terminal fields of other P B afferents, such as the infralimbic and insular cortices and the medial subdivision of the central nucleus, extend beyond the limits of their respective PB efferent cell populations. These projections may be less tightly reciprocal, or they may represent synapses located on distal dendrites of PB neurons with reciprocal connections. The functional significance of these different reciprocal connections is unknown.

M.M. MOGA ET AL. During the course of this study, we were able to make additional observations on the PB efferent connections, complementing a previous study from this laboratory (Fulwiler and Saper, '84). In particular, the superior lateral subnucleus was observed to project more widely in the forebrain than previously reported; besides projections to the ventromedial, paraventricular, and lateral hypothalamus, this subnucleus also innervates the retrochiasmatic area, the medial nucleus of the amygdala, and the preoptic part of the bed nucleus of the stria terminalis. The superior lateral input to the medial nucleus of the amygdala was previously described by Ottersen (W), who noted retrogradely labeled cells in the "dorsomedial extreme of the dorsal nucleus of the lateral lemniscus" following HRP injections restricted to the medial nucleus. Possibly, the cholecystokinin-like immunoreactive neurons in the superior lateral subnucleus, which were previously shown to project to the ventromedial hypothalamus (Fulwiler and Saper, '85; Zaborszky et al., '84), may project to the medial nucleus of the amgydala as well as to other terminal fields of this subnucleus. The PB projection to the bed nucleus of the stria terminalis is more complex than previously described. Fulwiler and Saper ('84) described retrogradely labeled neurons in the central lateral, dorsal lateral, and extreme lateral subnuclei after injections into the bed nucleus of the stria terminalis. This pattern is present after injections, such as R826 (Fig. 14), that include the parastrial nucleus. However, injections that include the lateral subnuclei of the BST subnuclei show additional retrograde labeling in the external lateral and waist PB subnuclei. The external lateral P B subnucleus also projects to the basomedial nucleus of the amygdala, (Fig. 3B), to the caudal lateral hypothalamus (Fig. 20H,I), and to the median preoptic nucleus of the hypothalamus (Fig. 19C). Furthermore, neurons in the inner and outer zones of the external lateral subnucleus project to different areas of the forebrain. For example, the caudal lateral hypothalamus, medial subdivision of the central nucleus, and substantia innominata receive input from cells in the inner zone, whereas the median preoptic nucleus, lateral subdivision of the central nucleus, and dorsal lateral BST subnucleus receive input from the outer zone. Neurons in these two zones may send collaterals to their different targets, but this has not been studied.

Correlationwith PB medullary afferents The present study demonstrates that the forebrain input to the P B is as extensive and as complexly organized as that from the medulla. The PB terminal fields of the forebrain afferents almost entirely overlap those of the medullary afferents, in some cases producing identical patterns of innervation in the PB, suggesting that particular forebrain afferents may modify specific types of ascending visceral information. For example, the central nucleus of the amygdala and the bed nucleus of the stria terminalis innervate the external lateral, central lateral, and waist subnuclei in a pattern nearly identical to that seen after medial nucleus of the solitary tract injections (Herbert et al., 'go), suggesting that the central nucleus of the amygdala and bed nucleus of the stria terminalis may modify information transmitted by ascending general visceral afferents. Similarly, the rostral insular cortex projects extensively to the rostral part of the ventral lateral subnucleus as well as to the medial and waist subnuclei; this pattern of innervation is

PARABRACHIAL FOREBRAIN AFFERENTS also observed after injections into the rostral part of the nucleus of the solitary tract (Herbert et al., '901, suggesting that this area of the cortex may modulate ascending gustatory information. Besides modifying the ascending visceral input, the forebrain afferents may also activate some of the autonomic responses associated with the PB. For example, electrical stimulation of the P B produces tachycardia, vasoconstriction, and an increase in blood pressure that is independent of any forebrain input (Mraovitch et al., '82). Stimulation of the central nucleus of the amygdala elicits a similar pressor response, presumably through its projections to the brainstem (Galeno and Brody, '83; Galeno et al., '84). Respiratory responses obtained from electrical stimulation of the central nucleus of the amygdala are also nearly identical to those elicited from the P B (Cohen, '77; Harper et al., '84; Holmes e t al., '87). Thus the forebrain pathways to the PB may be an important substrate for some of the autonomic responses elicited from forebrain cell populations.

ACKNOWLEDGMENTS This study was supported by NIH grant NS22835, the Brain Research Foundation, and American Heart Association grants 881120 and 850894. We thank David Standaert for the use of his material; David Cechetto and David Rye for helpful discussions; and Quan Ha, Sabina Herbert, and Debra Magnuson for their technical assistance.

LITERATURE CITED Amaral, D.G., and J.L. Price (1983) An air pressure system for the injection of tracer substances into the brain. J. Neurosci. Methods 9:3543. Barnard, J.W. (1936) A phylogenetic study of the visceral afferent areas associated with the facial, glossopharyngeal, and vagus nerves, and their fiber connections. The efferent facial nucleus. J. Comp. Neurol. 65:503602. Berk, M.L., and J.A. Finkelstein (1982) Efferent connections of the lateral hypothalamic area of the r a t An autoradiographic investigation. Brain Res. Bull. 8:511-526. Brennan, T.J., U.T. Oh, M.-N. Girardot, W.S. Ammons, and R.D. Foreman (1987) Inhibition of cardiopulmonary input to thoracic spinothalamic tract cells by stimulation of the subcoeruleus-parabrachialregion in the primate. J. Auton. Nervous System 1855-72. Cechetto, D.F., and C.B. Saper (1987) Evidence for a viscerotopic sensory representation in the cortex and thalamus in the rat. J. Comp. Neurol. 262r27-45. Cechetto, D.F., and C.B. Saper (1988) Neurochemical organization of the hypothalamic projection to the spinal cord in the rat. J. Comp. Neurol. 272:579404. Cechetto, D.F., D.G. Standaert, and C.B. Saper (1985) Spinal and trigeminal dorsal horn projections to the parabrachial nucleus in the rat. J. Comp. Neurol. 240r153-160. Chiba, T., and Y. Murata (1985) Afferent and efferent connections of the medial preoptic area in the rat: A WGA-HRP study. Brain Res. Bull. 14r261-272. Cohen, M.I. (1971) Switching of the respiratory phases and evoked phrenic responses produced by rostral pontine electrical stimulation. J. Physiol. 21 7t133-158. Conrad, L.C.A.. and D.W. Pfaff (1976) Efferents from medial basal forebrain and hypothalamus in the rat. I. An autoradiographic study of the medial preoptic area. J. Comp. Neurol. 769:185-220. de Olmos, J., G.F. Alheid, and C.A. Beltramino (1985) Amygdala. In G. Paxinos (ed.): The Rat Nervous System, Vol. 1,Forebrain and Midbrain. Orlando, FI,: Academic Press, pp. 223-334. de Olmos, J., H. Hardy, and L. Heimer (1978) The afferent connections of the main and accessory olfactory bulb formations in the rat: an experimental HRP-study. J. Comp. Neurol. 181.213-244. Feldman, J.L., and H. Gautier (1976) Interaction of pulmonary afferents and

659 pneumotaxic center in control of respiratory pattern in cats. J. Neurophysiol. 39r31-44. Fulwiler, C.E., and C.B. Saper (1984) Subnuclear organization of the efferent connections of the parahrachial nucleus in the rat. Brain Res. Rev. 7.229-259. Fulwiler, C.E., and C.B. Saper (1985) Cholecystokinin-immunoreactive innervation of the ventromedial hypothalamus in the r a t Possible substrate for autonomic regulation of feeding. Neurosci. Lett. 53r289-296. Galeno, T.M., and M..J. Brody (1983) Hemodynamic responses to amygdaloid stimulation in spontaneously hypertensive rats. Amer. J. Physiol. 245: R281-286. Galeno, T.M., G.W. Van Housen, and M.J. Brody (1984) Central amygdaloid nucleus lesion attenuates exaggerated hemodynamic responses to noise stress in the spontaneously hypertensive rat. Brain Res. 291:249-259. Gerfen, C.R., and P.E. Sawchenko (1984) An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: Immunohistochemical localization of an axonally transported plant lectin, Phaseolus vulgaris leucoagglutinin (PHA-L). Brain Res. 290:219-238. Gibson, A.R., D.I. Hansma, J.C. Houk, and F.R. Robinson (1984) A sensitive low artifact TMB procedure for the demonstration of WGA-HRP in the CNS. Brain Res. 298.235-241. Girardot, M.-K., T.J. Brennan, M.E. Martindale, and R.D. Foreman (1987) Effects of stimulating the subcoeruleus-parabrachial region on the nonnoxious and noxious responses of T,-T, spinothalamic tract neurons in the primate. Brain Res. 409.19-30. Gray, T.S., and D.J. Magnuson (1987a) Galanin-like immunoreactivity within amygdaloid and hypothalamic neurons that project to the midbrain central grey in rat. Xeurosci. Lett. 83r264-268. Gray, T.S., and D.J. Magnuson (1987b) Neuropeptide neuronal efferents from the bed nucleus of the stria terminalis and central amygdaloid nucleus to the dorsal vagal complex in the rat. J. Comp. Neurol. 262365-374. Grob, P., J. Buttner-Ennever, W. Iang, K. Akert, and A. Fah (1982) A comparison of the retrograde tracer properties of [li51]wheat germ agglutinin (WGA) with HRP after injection into the corpus callosum. Brain Res. 236:193-198. Grove, E.A. (1988) Efferent connections of the substantia innominata in the rat. J. Comp. Neurol. 277:347-364. Hammond, D.L., and H.K. Proudfit (1980) Effects of locus coeruleus lesions on morphine-induced antinociception. Brain Res. 188:79-81. Harper, R.M., R.C. Frysinger, R.B. Trelease, and J.D. Marks (1984) Statedependent alteration of respiratory cycle timing by stimulation of the central nucleus of the amygdala. Brain Res. 306:l-8. Hendry, S.H., E.G. Jones, J. Defelipe, D. Schmechel, C. Brandon, and P.C. Emson (1984) Neuropeptide-containing neurons of the cerehral cortex are also GABAergic. Proc. Natl. Acad. Sci. USA 81:6526-6530. Herhert, H., M.M. Moga, and C.B. Saper (1990) Connections of the parahrachial nucleus with the nucleus of the solitary tract and medullary reticular formation in the rat. J. Comp. Neurol. 293:540-580. Herrick, C.J. (1905) The central gustatory paths in the brains of bony fishes. J. Comp. Neurol. 153755456. Herrick, C.J. (1948) The Brain of the Tiger Salamander, Ambystoma tigrinurn. Chicago: University of Chicago Press. Holmes, H.R., H. Herbert, and C.B. Saper (1987) Parabrachial subnuclei in respiratory control. SOC.Neurosci. Abstr. 13r1586. Holstege, G., L. Meiners, and K. Tan (1985) Projections of the bed nucleus of the stria terminalis to the mesencephalon, pons, and medulla oblongata in the cat. Exp. Brain Res. 58r379-391. Hopkins, D.A., and G. Holstege (1978) Amygdaloid projections to the mesencephalon, pons and medulla oblongata in the cat. Exp. Brain Res. 32:529%547. Hopkins, D.A., J.H. McLean, and Y.Takeuchi (1981) Amygdalotegmental projections: light and electron microscopic studies utilizing anterograde degeneration and the anterograde and retrograde transport of horseradish peroxidase (HRP). In Y. Ben-Ari (ed): The Amygdaloid Complex. New York Elsevier/North-Holland Biomedical Press, pp. 133-147. Hosoya, Y. (1980) The distribution of spinal projection neurons in the hypothalamus of the rat, studied with the HRP method. Exp. Brain Res. 4039-87. Hosoya, Y., and M. Matsushita (1981) Brainstem projections from the lateral hypothalamic area in the rat, as studied with autoradiography. Neurosci. Lett. 24:lll-116.

660 Jackson, A,, and A.R. Crossman (1981) Basal ganglia and other afferent projections to the peribrachial region in the rat: A study using retrograde and anterograde transport of horseradish peroxidase. Neurosci. 6:15371549. Johnston, J.B. (1923) Further contributions to the study of the evolution of the forebrain. J. Comp. Neurol. 35:337-481. Krettek, J.E., and J.L. Price (1978) Amygdaloid projections to subcortical structures within the basal forebrain and brainstem in the rat and cat. J. Comp. Neurol. 178~225254. Loewy, A.D., C.U. Saper, and R.P. Baker (1979) Descending projections from the pontine micturition center. Brain Res. 172533-538. Luiten, P.G.M., G.J. ter Horst, H. Karst, and A.B. Steffens (1985) The course of paraventricular hypothalamic efferents to autonomic structures in medulla and spinal cord. Brain Res. 329:374-378. Lumb, B.M., and J.F.B. Morrison (1987) An excitatory influence of dorsolatera1 pontine structures on urinary bladder motility in the rat. Brain Res. 435:363-366. McDonald, A.J. (1982) Cytoarchitecture of the central amygdaloid nucleus of the rat. J. Comp. Neurol. 208:401-408. Millhouse, O.E. (1986) The intercalated cells of the amygdala. J. Comp. Neurol. 247:246-271. Milner, T.A., and V.M. Pickel (1986a) Neurotensin in the rat parabrachial region: Ultrastructural localization and extrinsic sources of immunoreactivity. J. Comp. Neurol. 247:326-343. Milner, T.A., and V.M. Pickel (1986b) Ultrastructural localization and afferent sources of substance P in the rat parabrachial region. Neuroscience 17:687-707. Moga, M.M., and T.S. Gray (1985a) Peptidergic efferents from the intercalated nuclei of the amygdala to the parabrachial nucleus. Neurosci. Lett. 61:13-18. Moga, M.M., and T.S. Gray (198513) Evidence for corticotropin releasing factor, neurotensin, and somatostatin in the neural pathway from the central nucleus of the amygdala to the parabrachial nucleus. J. Comp. Neurol. 241:275-284. Moga, M.M., K.M. Hurley-Gus, D.F. Cechetto, T.S. Gray, and C.B. Saper (1986) Distribution of forebrain afferents to the parabrachial nucleus in the rat. Neurosci. Abstr. 12:1054. Moga, M.M., C.B. Saper, and T.S. Gray (1989) The bed nucleus of the stria terminalis: Cytoarchitecture, immunohistochemistry and projection to the parabrachial nucleus in the rat. J. Comp. Neurol. 283:315-332. Moga, M.M., C.B. Saper, and T.S. Gray (1990) Neuropeptide organization of the hypothalamic projection to the parabrachial nucleus in the rat. J. Comp. Neurol. (this issue). Mraovitch, S., C. Iadecola, D.A. Ruggiero, and D.J. Reis (1985) Widespread reductions in cerebral blood flow and metabolism elicited by electrical stimulation of the parabrachial nucleus in rat. Brain Res. 341283-296. Mraovitch, S., M. Kumada, and D.J. Reis (1982) Role of the nucleus parabrachialis in cardiovascular regulation in cat. Brain Res. 23257-75. Norgren, R., and C.M. Leonard (1973) Ascending central gustatory pathways. J. Comp. Neurol. 150217-238. Ohman, L.E., and A.K. Johnson (1986) Lesions in lateral parabrachial nucleus enhance drinking to angiotensin I1 and isoproterenol. Am. J. Physiol. 251:R504-R509. Ottersen, O.P. (1 981) Afferent connections to the amygdaloid complex of the rat with some observations in the cat. 111. Afferents from the lower brain stem. J. Comp. Neurol. 202,335356. Price, J.L. (1981) The efferent projections of the amygdaloid complex in the rat, cat and monkey. In Y. Ben-Ari (ed): The Amygdaloid Complex. New York: Elsevier/North-Holland Biomedical Press, pp. 121-132. Price, J.L., and D.G. Amaral (1981) An autoradiographic study of the projections of the central nucleus of the monkey amygdala. J. Neurosci. 1:1242-1259. R o b , J.T.M., P.J.W. Juch, and J.D. van Willigen (1986a) Arrangement and connections of mesencephalic trigeminal neurons in the rat. Acta Anat. 127:7-15. Rokx, J.T.M., J.D. van Williegen, and P.G.W. Juch (1986b) Bilateral brainstem connections of the rat supratrigeminal region. Acta Anat. 127:16-21. Rye, D.B., C.B. Saper, H.J. Lee, and B.H. Wainer (1987) Pedunculopontine tegmental nucleus of the rat: Cytoarchitecture, cytochemistry, and some extrapyramidal connections of the mesopontine tegmentum. J. Comp. Neurol. 259:483-528.

M.M. MOGA ET AL. Sakanaka, M., T. Shibasaki, and K. Lederis (1986) Distribution and efferent projections of corticotropin-releasingfactor-like immunoreactivity in the rat amygdaloid complex. Brain Res. 382213-238. Saper, C.B. (1982) Reciprocal parabrachml-cortical connections in the rat. Brain Res. 24233-40. Saper, C.B., and D. Levisohn (1983) Afferent connections of the median preoptic nucleus in the rat: Anatomical evidence for a cardiovascular integrative mechanism in the anteroventral third ventricular (AV3V) region. Brain Res. 288.2-31. Saper, C.B., L.W. Swanson, and W.M. Cowan (1976) The efferent connections of the ventromedial nucleus of the hypothalamus of the rat. J. Comp. Neurol. 269:409-422. Saper, C.B., L.W. Swanson, and W.M. Cowan (1979) An autoradiographic study of the efferent connections of the lateral hypothalamic area in the rat. J. Comp. Neurol. 183~689-706. Satoh, K., N. Sbimizu, M. Tohyama, and T. Maeda (1978) Localization of the micturition reflex center at dorsolateral pontine tegmentum of the rat. Neurosci. Lett. 8~27-33. Schmechel, D.E., B.G. Vickrey, D. Fitzpatrick, and R.P. Elde (1984) GABAergic neurons of mammalian cerebral cortex: Widespread subclass defined by somatostatin content. Neurosci. Lett. 47:227-232. Schwaber, J.S., B.S. Kapp, G.A. Higgins, and P.R. Rapp (1982) Amygdaloid and basal forebrain direct connections with the nucleus of the solitary tract and the dorsal motor nucleus. J. Neurosci. 21424-1438. Shapiro, R.E., and R.R. Miselis (1985) The central neural connections of the area postrema of the rat. J. Comp. Neurol. 234:344-364. Shimada, S., S. Inagaki, Y. Kubota, N. Ogawa, T. Shibasaki, and H. Takagi (1989) Coexistence of peptides (corticotropin releasing factorheurotensin and substance P/somatostatin) in the bed nucleus of the stria terminalis and central amygdaloid nucleus of the rat. Neuroscience 30:377-384. Shipley, M.T., and M.S. Sanders (1982) Special senses are really special: Evidence for a reciprocal, bilateral pathway between insular cortex and nucleus parabrachialis. Brain Res. Bull. 8:493-501. Simerly, R.B., and L.W. Swanson (1988) Projections of the medial preoptic nucleus: A Phaseolus vulgaris leucoagglutinin anterograde tract-tracing study in the rat. J. Comp. Neurol. 270:209-242. Simerly, R.B., L.W. Swanson, and R.A. Gorski (1984) Demonstration of a sexual dimorphism in the distribution of serotonin-immunoreactive fibers in the medial preoptic nucleus of the rat. J. Comp. Neurol. 225:151- 166. Swanson, L.W. (1976) An autoradiographic study of the efferent connections of the preoptic region in the rat. J. Comp. Neurol. 167:227-256. Swanson, L.W. (1977) Immunohistochemical evidence for a neurophysincontaining autonomic pathway arising in the paraventricular nucleus of the hypothalamus. Brain Res. 128:346-353. Swanson, L.W., and W.M. Cowan (1979) The connections of the septal region in the rat. J. Comp. Neurol. 186:621-656. Swanson, L.W., and H.G.J.M. Kuypers (1980) A direct projection from the ventromedial nucleus and retrochiasmatic area of the hypothalamus to the medulla and spinal cord of the rat. Neurosci. Lett. 17:307-312. Takeuchi, Y., G.V. Allen, and D.A. Hopkins (1985) Transnuclear transport and axon collateral projections of the mammillary nuclei in the rat. Brain Res. Bull. 14r453-468. Takeuchi, Y., and D.A. Hopkins (1984) Light and electron microscopic demonstration of hypothalamic projections to the parabrachial nuclei in the cat, Neurosci. Lett. 46:53-58. Takeuchi, Y., J.H. McLean, and D.A. Hopkins (1982) Reciprocal connections between the amygdala and parabrachial nuclei: Ultrastructural demonstration by degeneration and axonal transport of horseradish peroxidase in the cat. Brain Res. 239:583-588. ter Horst, G.J., and P.G.M. Luiten (1986) The projections of the dorsomedial hypothalamic nucleus in the rat. Brain Res. Bull. 16231-248. Terreberry, R.R., and E.J. Neafsey (1987) The rat medial frontal cortex projects directly to autonomic regions of the brainstem. Brain Res. Bull. 19:639449. van der Kooy. D., L.Y. Koda, J.F. McGinty, C.R. Gerfen, and F.E. Bloom (1984) The organization of projections from the cortex, amygdala, and hypothalamus to the nucleus of the solitary tract in rat. J. Comp. Neurol. 224:l-24. Veening, J.G., L.W. Swanson, and P.E. Sawchenko (1984) The organization of projections from the central nucleus of the amygdala to brainstem sites involved in central autonomic regulation: A combined retrograde transport-immunohistochemical study. Brain Res. 303:337-357.

PARABRACHIAL FOREBRAIN AFFERENTS Veening, J.G., S. Te Lie, P. Posthuma, L.M.G. Geeraedts, and It. Nieuwenhuys (1987) A topographical analysis of the origin of some efferent projections from the lateral hypothalamic area in the rat. Neuroscience 22537-551. von Euler, C., I. Martilla, J.E. Remmers, and T. Trippenbach (1976) Effects of lesions in the parabrachial nucleus on the mechanisms for central and reflex termination of inspiration in the cat. Acta Physiol. Scand. 96:324337. Yasui, Y., K. Itoh, M. Takada, A. Mitani, T. Kaneko, and N. Mizuno (1985) Direct cortical projections to the parahrachial nucleus in the cat. J. Comp. Neurol. 234:77-86.

66 1 Yasui, Y., C.D. Breder, C.B. Saper, and D.F. Cechetto (1990) Autonomic responses and efferent pathways from the insular cortex in the rat. In preparation. Zabbrszky, L., M.C. Beinfeld, M. Palkovits, and L. Heimer (1984) Brainstem projection to the hypothalamic ventromedial nucleus in the rat: A CCK-containing long ascending pathway. Brain Res. 303225-231. Zardetto-Smith, A.M., and T.S. Gray (1987) Efferent projections of the nucleus of the solitary tract to the amygdala, bed nucleus of the stria terminalis and subfornical organ in the rat examined using the PHA-L anterograde tracing method. SOC.Neurosci. Abstr. 13:386.