Organization of neural inputs to the suprachiasmatic nucleus in the rat

Organization of Neural Inputs to theSuprachiasmatic Nucleus in the Rat

MARGARET M. MOGA1* AND ROBERT Y. MOORE2

1Department of Anatomy, Indiana University School of Medicine,Terre Haute, Indiana 47809

2Center for Neuroscience and Departments of Psychiatry, Neurology, and Neuroscience,University of Pittsburgh, Pittsburgh, Pennsylvania 15261

ABSTRACTThe circadian timing of the suprachiasmatic nucleus (SCN) is modulated by its neural

inputs. In the present study, we examine the organization of the neural inputs to the rat SCNusing both retrograde and anterograde tracing methods. After Fluoro-Gold injections into theSCN, retrogradely labeled neurons are present in a number of brain areas, including theinfralimbic cortex, the lateral septum, the medial preoptic area, the subfornical organ, theparaventricular thalamus, the subparaventricular zone, the ventromedial hypothalamicnucleus, the posterior hypothalamic area, the intergeniculate leaflet, the olivary pretectalnucleus, the ventral subiculum, and the median raphe nuclei. In the anterograde tracingexperiments, we observe three patterns of afferent termination within the SCN thatcorrespond to the photic/raphe, limbic/hypothalamic, and thalamic inputs. The median rapheprojection to the SCN terminates densely within the ventral subdivision and sparsely withinthe dorsal subdivision. Similarly, areas that receive photic input, such as the retina, theintergeniculate leaflet, and the pretectal area, densely innervate the ventral SCN but provideonly minor innervation of the dorsal SCN. A complementary pattern of axonal labeling, withlabeled fibers concentrated in the dorsal SCN, is observed after anterograde tracer injectionsinto the hypothalamus and into limbic areas, such as the ventral subiculum and infralimbiccortex. A third, less common pattern of labeling, exemplified by the paraventricular thalamicafferents, consists of diffuse axonal labeling throughout the SCN. Our results show that theSCN afferent connections are topographically organized. These hodological differences mayreflect a functional heterogeneity within the SCN. J. Comp. Neurol. 389:508–534,1997. r 1997 Wiley-Liss, Inc.

Indexing terms: circadian rhythm; intergeniculate leaflet; paraventricular thalamus; hypothalamus

The suprachiasmatic nucleus (SCN) is the anatomicallocus of the mammalian circadian clock (for reviews, seeVan den Pol and Dudek, 1993; Moore, 1994; Morin, 1994).One of the more remarkable findings in circadian biologywas the discovery that the circadian rhythmicity of theSCN persists in vitro in both hypothalamic slice and cellculture preparations (Green and Gillette, 1982; Groos andHendricks, 1982; Shibata et al., 1982; Earnest and Sladek,1986; Murakami et al., 1991; Mirmiran et al., 1995; Welshet al., 1995). In hypothalamic slices, the firing rate ofindividual SCN neurons varies over the course of approxi-mately 24 hours (i.e., circadian), with a peak in firingoccurring during the animal’s subjective day (for review,see Gillette, 1991). In SCN cell cultures, the neuropeptidesvasopressin and vasoactive intestinal peptide (VIP) show acircadian pattern of release, with periods ranging from 22hours to 27 hours (Murakami et al., 1991; Watanabe et al.,1993; Shinohara et al., 1994). The autonomy of the SCN

circadian pacemaker is further evident in SCN transplantstudies (for review, see Ralph and Lehman, 1991). In thesestudies, the circadian rhythm of locomotor activity isabolished with lesions of the SCN; this rhythm is thenrestored by SCN transplants placed in the third ventricle(Lehman et al., 1987; Aguilar-Roblero et al., 1992; Grif-fioen et al., 1993). The period of the restored rhythm isdetermined by the donor SCN tissue (Ralph et al., 1990).These findings demonstrate that the circadian periodicityof the SCN is an intrinsic phenomenon that does notdepend on inputs from brain areas outside the SCN.

Grant sponsor: NIH; Grant number: NS-16304.*Correspondence to: Dr. Margaret M. Moga, 135 Holmstedt Hall, Indiana

State University School of Medicine, Terre Haute, IN 47809-9989. E-mail:[email protected]

Received 10 April 1997; Revised 18 July 1997; Accepted 28 July 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 389:508–534 (1997)

r 1997 WILEY-LISS, INC.

What, then, is the role of the neural inputs to the SCN?Only three SCN neural inputs have been studied in depth,namely from the retina, the midbrain raphe, and theintergeniculate leaflet (IGL). The primary function of theretinal input is to entrain the intrinsic circadian rhythm ofthe SCN to the prevailing light-dark cycle (for reviews, seeMorin, 1994; Moore, 1995, 1996). The serotonergic input,originating from neurons in the midbrain raphe, modu-lates the circadian pacemaker’s response to light (Selim etal., 1993; Rea et al., 1994; for review, see Morin, 1994). TheIGL appears to mediate nonphotic entrainment of circa-dian rhythms (Johnson et al., 1988a; Edgar et al., 1991;Janik and Mrosovsky, 1994; Moore and Card, 1994). Thefunctional specificity of these neural inputs suggests thateach of the SCN afferents may play a unique modulatoryrole in circadian timing.

The SCN receives neural input from a number of othersources. After retrograde tracer injections into the SCN,labeled cells have been observed in the medial preopticarea, the subparaventricular zone, the paraventricularthalamus, the ventromedial hypothalamic nucleus (VMH),the arcuate nucleus of the hypothalamus, the lateralseptal nucleus, the substantia innominata, the ventralsubiculum, the pretectal area, the central gray, the pedun-culopontine nucleus, the parabrachial nucleus, and thezona incerta (Moore et al., 1979; Pickard, 1982; Bina et al.,1993; Mikkelsen and Vrang, 1994). There has been antero-grade confirmation of some, but not all, of these inputs tothe SCN (Garris, 1979; Silverman and Oldfield, 1984;Oldfield and Silverman, 1985; Simerly and Swanson,1988; Staiger and Nurnberger, 1991; Canteras and Swan-son, 1992; Bina et al., 1993; Mikkelsen and Vrang, 1994;

Moga et al., 1995). Several neural inputs to the SCN,notably from the infralimbic cortex, subfornical organ,dorsomedial hypothalamic nucleus, and spinal cord, havebeen described with anterograde tracing but not withretrograde tracing methods; thus, their precise origins areunknown (Swanson and Lind, 1986; Cliffer et al., 1991;Hurley et al., 1991; Thompson et al., 1996).

The SCN afferents may be topographically organizedalong previously defined subnuclear boundaries, but thishas not been systematically examined. The rat SCN can bedivided into two major subdivisions based on cytoarchitec-ture and neuropeptide immunoreactivity: 1) a ventralsubdivision with VIP- and gastrin-releasing peptide-immunoreactive cells (Van den Pol, 1980; Moore, 1983; Vanden Pol and Tsujimoto, 1985; Speh et al., 1994; Moore,1996) and 2) a dorsal subdivision with vasopressin-immunoreactive cells. Both the retina and the IGL senddense projections to the ventral SCN subdivision, withlittle innervation of the dorsal subdivision (Card andMoore, 1982; Harrington et al., 1985; Johnson et al.,1988b; Levine et al., 1991; Morin et al., 1992; Janik andMrosovsky, 1994). The midbrain raphe nuclei innervatethe entire SCN but with a ventral predominance (Azmitiaand Segal, 1978; Moore et al., 1978). Axons from thepretectum are clustered along the lateral border of theSCN and terminate within the ventral and lateral portionsof the nucleus (Mikkelsen and Vrang, 1994). The infralim-bic cortex projects solely to the dorsal SCN (Hurley et al.,1991). In contrast, some SCN afferent cell populations,such as the paraventricular thalamic nucleus and thesubparaventricular zone, innervate the entire SCN in adiffuse manner (Watts et al., 1987; Moga et al., 1995).

Abbreviations

3V third ventricleac anterior commissureAcb accumbens nucleusAHA anterior hypothalamic areaAPT anterior preoptic nucleusArc arcuate nucleus of the hypothalamusCA1 CA1 field of hippocampusCeL central nucleus of the amygdala, lateralCeM central nucleus of the amygdala, medialCg cingulate cortexCG central grayCnf cuneiform nucleusCP caudate putamenDB nucleus of the diagonal bandDG dentate gyrusDLG dorsal lateral geniculatedm dorsomedial part of the SCNDMH dorsomedial hypothalamic nucleusDR dorsal rapheDT dorsal tegmental nucleusEnt entorhinal cortexFF fields of Forelfr fasciculus retroflexusGP globus pallidusic internal capsuleIC inferior colliculusICj islands of CallejaIGL intergeniculate leafletIL infralimbic cortexLDT laterodorsal tegmental nucleusLHA lateral hypothalamic areaLS lateral septumLV lateral ventricleMD mediodorsal thalamic nucleusMG medial geniculatemlf medial longitudinal fasciculus

MnPO median preoptic nucleusMnR median rapheMo5 motor trigeminal nucleusMPO medial preoptic areaMPT medial pretectal areaMT medial terminal nucleusND nucleus of Darkschewitschopt optic tractOPT olivary pretectal nucleusPB parabrachial nucleusPHA posterior hypothalamic areaPPN pedunculopontine nucleusPPT posterior pretectal nucleusPT paratenial thalamic nucleusPVH paraventricular hypothalamic nucleusPVT paraventricular thalamic nucleusRe reuniens thalamic nucleusRP raphe pontisSC superior colliculusSCN suprachiasmatic nucleusscp superior cerebellar peduncleSFO subfornical organSI substantia innominatasm stria medullarisSO supraoptic nucleussox supraoptic commissureSPZ subparaventricular zoneti tractus infundibularisTMN tuberomammillary nucleusTT tenia tectavl ventrolateral part of the SCNVLG ventral lateral geniculateVMH ventromedial hypothalamic nucleusVSub ventral subiculumVTg ventral tegmental nucleusZI zona incerta

SCN AFFERENT CONNECTIONS 509

The aim of the present experiments is a comprehensiveanalysis of the SCN afferent connections using both antero-grade and retrograde tracing methods. In the first set ofexperiments, the retrograde tracer Fluoro-Gold (FG) wasinjected iontophoretically into the SCN to identify possiblesources of afferent input to the SCN. In the second set ofexperiments, the anterograde tracers Phaseolus vulgaris-leucoagglutinin (PHA-L) and biotinylated dextran (BDX)were injected into 18 areas that were identified in theretrograde experiments as potential SCN afferent cellpopulations. The purpose of the anterograde experimentswas twofold: to substantiate the retrograde findings and todelineate the terminal fields of the SCN afferents. Prelimi-nary findings of this study were reported previously (Mogaand Moore, 1994).

MATERIALS AND METHODS

Adult male Sprague Dawley rats weighing 250–350 gwere anesthetized with chloral hydrate (40–50 mg/kg).The experimental protocols in this study were approved bythe Animal Care Committee of the University of Pitts-burgh Medical Center.

Fluoro-Gold injections. Fluoro-Gold (FG) (2% in 0.1M cacodylate buffer, pH 8.5; Fluorochrome, Inc., Engle-wood, CO; Schmued and Heimer, 1990) was stereotaxicallyiontophoresed into the SCN of 11 rats. The inner tipdiameter of the glass micropipette was 20–25 µm. Theinjection parameters were 5 µA of positive current every 10seconds for 3–5 minutes. The micropipette was left in placefor 10–15 minutes after the injection. After a 14-daysurvival period, the animals were deeply anesthetizedwith chloral hydrate (150 mg/kg) and perfused through theascending aorta with 150 ml of heparinized saline; fol-lowed by 500 ml of 4% paraformaldehyde in 0.1 M phos-phate buffer, pH 7.4; and then 50 ml of 30% sucrose in 0.1M phosphate buffer, pH 7.4. The brains were removed,postfixed if necessary, and allowed to sink overnight in the30% sucrose solution. Brains were cut into 30-µm sectionson a freezing microtome.

Aone-of-six series of sections (a one-of-two series throughthe injection site and the SCN) was preincubated for 1hour in a 0.01 M phosphate-buffered saline solution (PBS),pH 7.4, with 2% normal donkey serum (NDS) and 0.3%Triton X-100 (TX). The sections were then incubated in theprimary antibody [anti-FG immunoglobulin (IgG); Chemi-con, Temecula, CA; diluted 1:15,000 in PBS-NDS-TX;Chang et al., 1990] with gentle agitation for 48 hours at4°C. Next, the sections were rinsed in PBS, incubated inthe secondary antibody (donkey anti-rabbit IgG; Jackson,West Grove, PA; diluted 1:200 in PBS-NDS-TX) for 1–2hours at room temperature, rinsed again, and reacted withavidin-biotin complex (ABC) reagent (Elite ABC kit; VectorLaboratories, Burlingame, CA) for 1–2 hours. After severalrinses in PBS, the sections were reacted in a solution of0.05% 3,38-diaminobenzidine tetrahydrochloride (DAB;Sigma, St. Louis, MO) 20.01% H2O2 in 0.1 M Tris buffer,pH 7.4, for 10 minutes. The sections were rinsed again inPBS, mounted on gel-coated slides, allowed to dry, rehy-drated, placed in 0.4% osmium tetroxide in PBS for 30–60seconds to intensify staining, dehydrated through alcoholsand xylene, and then coverslipped with Permount (FisherScientific, Pittsburgh, PA).

Phaseolus vulgaris-leucoagglutinin injections. A2.5% solution of PHA-L (Vector Laboratories) in a glassmicropipette (6–12 µm inner tip diameter) was stereotaxi-cally iontophoresed into select SCN afferent cell popula-tions. The injection parameters were 5 µA of positive directcurrent delivered in 10-second pulses for 10-20 minutes.The micropipette was left in place for 10 minutes followingthe injection. After a 14-day survival period, the animalswere deeply anesthetized and then perfused with 150 ml ofheparinized saline; followed by 500 ml of 4% paraformalde-hyde-0.05% glutaraldehyde in 0.1 M phosphate buffer, pH7.4; and then 50 ml of 30% sucrose in 0.1 M phosphatebuffer, pH 7.4.

The immunohistochemical procedure for PHA-L wasidentical to that used for FG visualization, except for thefollowing reagents: The primary antibody was goat anti-PHA (Vector Laboratories) diluted 1:15,000 in PBS-NDS-TX. The secondary antibody was donkey anti-goat IgG(Jackson) diluted 1:200 in PBS-NDS-TX.

Biotinylated dextran injections. A 5% solution ofBDX (Molecular Probes, Eugene, OR) that was freshlydissolved in double-distilled water was pressure-injectedinto select SCN afferent cell populations with a glassmicropipette (20–30 µm inner tip diameter) attached to a1-µl Hamilton syringe. After a survival period of 12–15days, the animals were deeply anesthetized with chloralhydrate and perfused with 150 ml of heparinized saline;followed by 500 ml of 4% paraformaldehyde in 0.1 Mphosphate buffer, pH 7.4; and then 50 ml of 30% sucrose inphosphate buffer.

A one-of-six series of 30-µm sections (a one-of-two seriesthrough the injection site and the SCN) was reacted withABC solution for 1–2 hours at room temperature. Afterseveral rinses in PBS, the sections were reacted in asolution of 0.05% DAB–0.01% H2O2 in 0.1 M Tris buffer, pH7.4, for 10 minutes. The sections were rinsed, mounted,and coverslipped as described for FG.

Sections were plotted with a camera lucida and/orphotographed with an Olympus Vanox microscope camerasystem (Tokyo, Japan). After plotting, the slides wereimmersed in xylene, and the coverslips were removed. Thesections were then counterstained with either neutral redor cresyl violet. Additional cytoarchitectonic details wereadded as necessary. The cytoarchitectonic atlases of Paxi-nos and Watson (1986) and Swanson (1992) were used forreference.

RESULTS

SCN cytoarchitecture

The cytoarchitecture of the SCN has been described inmany mammalian species, including rat, hamster, human,mouse, guinea pig, opossum, and cat (Van den Pol, 1980;Lydic et al., 1982; Guldner, 1983, 1985; Card and Moore,1984; Braak and Braak, 1987; Swanson, 1987; Cassone etal., 1988; Johnson et al., 1988b; Simerly, 1995). In agree-ment with these studies, we define the SCN as the clusterof small, densely packed neurons located in the rostral,ventral hypothalamus (Fig. 1). The rat SCN is borderedventrally by the optic chiasm and supraoptic commissures,and medially by the tractus infundibularis (Van den Pol,1980) and third ventricle. The lateral and dorsal bound-aries of the rat SCN are less distinct, but they aregenerally agreed upon (Van den Pol, 1980; Lydic et al.,

510 M.M. MOGA AND R.Y. MOORE

1982; Guldner, 1983, 1985; Card and Moore, 1984; Swan-son, 1987; Johnson et al., 1988b; Simerly, 1995). In each ofthese neuroanatomical studies as well as in the presentstudy, the dorsal and lateral borders of the SCN aredefined as the zone of transition between the small,densely packed cells of the SCN and the larger, morewidely spaced cells of the anterior hypothalamic area (Fig.1). To aid in the interpretation of our anterograde results,we have included a camera lucida drawing of a Nissl-stained section through the SCN (Fig. 1) that illustrateshow we have drawn the boundaries of the SCN for each ofour anterograde experiments.

The anterior hypothalamic area immediately surround-ing the SCN is often called the peri-SCN area or peri-SCNregion (Watson et al., 1995), and the area extending fromthe SCN to the paraventricular nucleus has been definedas the subparaventricular zone (Watts, 1991). The bound-aries of the peri-SCN area and the subparaventricularzone are indistinct in Nissl-stained sections, but they canbe approximated with immunohistochemistry and/or neu-ronal tract tracing (Watts, 1991; Watson et al., 1995).

Retrograde labeling experiments

To identify potential SCN afferent cell populations, weinjected the retrograde tracer FG into the SCN of 11animals. All of the FG injection sites in these experimentsare small and well localized, but none was restricted

entirely to the SCN. Injection sites for five representativeexperiments are illustrated in Figure 2. For each injectionsite in Figure 2, the stippled area depicts the extent of thedarkly staining inner core of the FG injection site; theneuropil in this zone has a distinct granular appearance.The cross-hatched areas in Figure 2 depict the diffusespread of FG from the centers of each injection site; thisouter zone is characterized by its many labeled glia. Weinterpret the darkly staining, granular core as the zone ofmaximal tracer uptake for the following reasons. 1) In aprevious set of FG experiments (Moga and Saper, 1994),we found no retrograde neuronal labeling in cases withvery small FG injection sites; these injection sites lacked agranular inner core and showed only diffuse glial labeling.2) Retrogradely labeled cells are most numerous in anSCN afferent cell population if the inner zone (i.e., darklystaining core) includes the SCN terminal field for that cellpopulation. For example, the granular, darkly stainingcore zone of the injection site in experiment R159 (Fig. 2)largely avoids the dorsal and lateral portions of the SCN.In this case, retrogradely labeled cells are scarce in limbicareas, such as the infralimbic cortex and lateral septalnucleus, that project to the dorsolateral SCN. In contrast,many retrogradely labeled cells are present in the infralim-bic cortex and lateral septal nucleus of experiment R160;the core zone in this experiment extends into the dorsolat-eral SCN (Fig. 2).

Fig. 1. A: Photomicrograph of a 30-µm-thick, Nissl-stained (neutral red), coronal section through amidrostrocaudal level of the suprachiasmatic nucleus (SCN; experiment R142). B: Camera lucidadrawing of the coronal section in A illustrating the cytoarchitectural boundaries of the SCN and adjacentstructures. For abbreviations, see list. Scale bar 5 100 µm.


Because of the difficulty of injecting the SCN in itsentirety without involving adjacent structures, essentiallyall of our FG experiments show 1) some labeling in

non-SCN afferent cell populations and/or 2) a lack oflabeling in SCN afferent cell populations that project tosubdivisions not included in an injection site (e.g., thedorsal and lateral portions of the SCN in experimentR159). The retrogradely labeled cell populations illus-trated in Figures 3–5 have been confirmed as SCN afferentcell populations based on the results of our anterogradetracing experiments and the experiments of other investi-gators (Watts et al., 1987; Simerly and Swanson, 1988;Mikkelsen and Vrang, 1994). The following descriptionspecifically refers to representative experiment R160. Inthis experiment, the injection site includes all of the SCN,with relatively little involvement of the supraoptic commis-sures or the anterior hypothalamic area (Fig. 2). Followingthe description of experiment R160, we describe the addi-tional retrograde labeling found in experiments with sub-stantial injection site involvement of the anterior hypotha-lamic area (experiment 93–87) or the supraopticcommissures (experiment R159).

Experiment R160. At rostral levels, a few retro-gradely labeled cells are present in layers 5 and 6 of theinfralimbic cortex. Many small, ovoid cells are retro-gradely labeled in the ventrolateral part of the lateralseptal nucleus (Fig. 3A). Scattered cells are found in themedial septal nucleus, the nucleus of the diagonal band,and the substantia innominata. Retrogradely labeled cellsare present in the rostral part of the median preopticnucleus, capping the vascular organ of the lamina termina-lis; these labeled cells continue ventrally and laterally intothe anteroventral periventricular nucleus and medial pre-optic area. At the level of the anterior commissure decussa-tion, only a few cells are found within the median preopticnucleus but many cells are present in the medial preopticarea (Fig. 3B). The paraventricular nucleus of the thala-mus contains many labeled cells, particularly in the ros-tral and dorsal portions of the nucleus (Fig. 3C). Retro-gradely labeled cells in the subfornical organ are locatedalong the periphery, surrounding the central core (Fig.3D). A few labeled cells are observed in the contralateralSCN. The retrochiasmatic area, immediately caudal to theSCN, contains retrogradely labeled neurons.

A small, dense cluster of cells is present in the subpara-ventricular zone, immediately ventral to the paraventricu-lar hypothalamic nucleus (Fig. 4A). Retrograde labeling isobserved in the rostral part of the zona incerta, immedi-ately dorsal and lateral to the paraventricular hypotha-lamic nucleus. Large numbers of labeled cells are found inthe VMH, particularly in the anterior and dorsal parts ofthe nucleus (Fig. 4B). A few cells in the arcuate nucleus areretrogradely labeled (Fig. 4B). The dorsomedial hypotha-lamic nucleus shows retrograde labeling in its rostral anddiffuse portions. Labeled cells are scattered throughoutthe lateral hypothalamic area. At caudal levels of thehypothalamus, a few magnocellular cells are retrogradelylabeled in the tuberomammillary nucleus. The ventralpremammillary nucleus and posterior periventricularnucleus contain a few labeled cells. A group of labeled

Fig. 2. Camera lucida drawings of SCN injection sites in fiverepresentative Fluoro-Gold (FG) experiments (R159, R160, R161,93-87, and 93-110). Two zones of labeling can be distinguished withineach FG injection site: a dark-staining inner zone (indicated bystippling) and a light-staining outer zone (indicated by cross-hatching). For abbreviations, see list.


neurons is located in the posterior hypothalamic area.Within the caudal thalamus, a tight cluster of retrogradelylabeled cells is present in the IGL (Fig. 4C). A few scattered

neurons are found in the ventral lateral geniculate nucleus.In the pretectum, retrogradely labeled cells are located inthe shell region of the olivary pretectal nucleus (Fig. 4D),

Fig. 3. Photomicrographs of retrogradely labeled cells in experi-ment R160 with an FG injection site centered in the SCN. A: A smallnumber of labeled neurons in the lateral septum (LS). B: Manyretrogradely labeled cells in the medial preoptic area (MPO), with a

few cells located in the median preoptic nucleus (MnPO) beneath theanterior commissure (ac). C: Labeled neurons in the rostral, bilobatepart of the paraventricular thalamic nucleus (PVT). D: Labeled cells inthe annular portion of the subfornical organ (SFO). Scale bar 5 100 µm.


Fig. 4. Photomicrographs of retrogradely labeled cells in experi-ment R160 with an FG injection site centered in the SCN. A: A clusterof labeled cells in the subparaventricular zone (SPZ; indicated byarrow). B: Retrogradely labeled cells in the ventromedial (VMH) andarcuate (Arc) nuclei of the hypothalamus. C: Labeled cells in the

intergeniculate leaflet (IGL; indicated by arrow). D: Two retrogradelylabeled neurons (arrowheads) along the perimeter of the olivarypretectal nucleus (OPT). For other abbreviations, see list. Scale bar 5100 µm.


with a few cells also present in the medial pretectal areaand, to a lesser extent, in the posterior pretectal nucleus.In the ventral subiculum, retrogradely labeled cells areconcentrated in the medial, distal portion with a fewlabeled cells also present in the lateral, proximal portion.

A small number of retrogradely labeled neurons islocated in the lateral dorsal subdivision of the central gray(for subdivisions, see Paxinos and Watson, 1986). Labeledneurons are present in the parabigeminal nucleus. In thedorsal raphe nucleus, retrogradely labeled cells are concen-trated in the lateral cell group, with only a few scatteredcells in the central cell groups (Fig. 5A). Labeled cells inthe lateral cell group of the dorsal raphe nucleus mergecaudally with cells in the laterodorsal tegmental nucleus.Many retrogradely labeled cells are located in the mid-brain median raphe and pontine median raphe nuclei (Fig.5B). A few cells in the locus coeruleus, the parabrachialnucleus (specifically, the central lateral subnucleus), andthe ventrolateral medulla are retrogradely labeled.

Experiment 93-87. The injection site in experiment93-87 encompasses an area larger than that in experimentR160, and shows considerable spread into the anteriorhypothalamic area (Fig. 2). In this case, large numbers oflabeled cells are observed in the infralimbic cortex as wellas in the prelimbic, dorsal peduncular, and entorhinalcortices. Retrogradely labeled cells are abundant in sev-eral other limbic areas, including the lateral septum, theventral subiculum, and the medial amygdaloid nucleus.

The pattern of retrograde labeling in the hypothalamus ofexperiment 93-87 is similar to that observed in experimentR160 but the number of hypothalamic labeled cells isincreased, particularly in the dorsomedial, arcuate, tuber-omammillary, and posterior periventricular hypothalamicnuclei and in the lateral and posterior hypothalamic areas.Retrogradely labeled cells are abundant in the medialpretectal area, the posterior pretectal nucleus, and theprecommissural nucleus; these areas show scarce labelingin experiment R160.

Experiment R159. The injection site in experimentR159 is small and centered in the rostral part of theventrolateral SCN. This injection site shows considerableinvolvement of the underlying supraoptic commissures(Fig. 2). The distribution of retrograde labeling in thetelencephalon of experiment R159 is virtually identical tothat observed in experiment R160, with the followingexceptions. In experiment R159, many lightly labeledneurons are present in the medial part of the olfactorytubercle. In the amygdala, a discrete cluster of retro-gradely labeled cells is observed in the posterodorsaldivision of the medial nucleus. Retrograde labeling ispresent in the subgeniculate nucleus, located immediatelyventral to the ventral lateral geniculate nucleus. Labeledcells are numerous in the anterior hypothalamic nucleus.

Retrograde labeling in the brainstem is greatly in-creased in experiment R159 compared with experimentR160. Increased numbers of labeled cells are noted in the

Fig. 5. Photomicrographs of retrogradely labeled cells in experiment R160 with an FG injection sitecentered in the SCN. A: Three labeled cells (arrowheads) in the lateral cell group of the dorsal raphenucleus (DR). B: Labeled neurons (two indicated by arrowheads) in the median raphe nucleus (MnR).Scale bar 5 100 µm.


central gray, the superior colliculus, the parabigeminalnucleus, the pedunculopontine nucleus, the dorsal raphenucleus, the laterodorsal tegmental nucleus, the cunei-form nucleus, and the parabrachial nucleus. Scatteredlarge, multipolar cells are present in the deep dorsal hornof the spinal cord; these cells are most prevalent at cervicallevels. Small, labeled cells are also found in the intermedi-ate zone of the cervical spinal cord.

Anterograde labeling experiments

In 44 animals, an anterograde tracer (either PHA-L orBDX) was injected into one of 18 different brain areas thatwere identified in the retrograde tracing experiments aspotential SCN afferent cell populations. Both PHA-L andBDX are sensitive anterograde tracers, and both produceGolgi-like staining of axons and terminal boutons withlittle uptake by fibers-of-passage (Gerfen and Sawchenko,1984; Wouterlood and Groenewegen, 1985; Brandt andApkarian, 1992; Veenman et al., 1992; Rajakumar et al.,1993). We chose BDX as the anterograde tracer for most ofour anterograde experiments because of its quick andsimple histochemical visualization with an ABC reagent.In contrast, PHA-L requires two antibody incubations inaddition to the ABC reaction. Because BDX is a relativelynew tracer, we provide a short account of our findingsregarding its use and efficacy.

With small injection volumes of BDX delivered slowly(20–80 nl at a rate of 10 nl every 2–3 minutes), we obtainfairly well-defined injection sites (Fig. 6), although theseinjection sites are not as well delineated as those withPHA-L. BDX injection sites are composed of granular,dark-staining neuropil with many labeled cells and cellprocesses. In agreement with previous studies (Veenmanet al., 1992; Rajakumar et al., 1993; Naito and Kita, 1994),we observe both anterograde and retrograde labeling withBDX. The anterograde labeling in our BDX experiments iscomparable in morphological detail to that in our PHA-L

experiments. The retrograde labeling is much less thanwhat we observe after injecting identical areas with FG orwheat germ agglutinin-horseradish peroxidase (WGA-HRP). Furthermore, the retrogradely labeled cells arelargely restricted to afferent cell populations in closeproximity to the injection site (e.g., cells in the VMH areretrogradely labeled after a BDX injection into the arcuatenucleus; Fig. 6A). We find little evidence for BDX uptakeby undamaged fibers-of-passage. In one experiment with aBDX injection placed directly in the optic chiasm, weobserve very few labeled fibers in the dorsal lateralgeniculate nucleus (1–2 glomerular arrays per section),indicating that very little uptake by fibers-of-passage hasoccurred.

In each of our anterograde experiments involving anSCN afferent cell population, axonal labeling is presentbilaterally in the SCN with an ipsilateral predominance.All photomicrographs and camera lucida drawings in thefollowing descriptions depict the ipsilateral SCN. In thecamera lucida drawings of representative anterogradeexperiments (Figs. 7, 8, 10–12), the BDX and PHA-L fiberswithin the SCN are represented 1:1. In some cases withdense axonal labeling outside the SCN, the pattern ofextra-SCN fiber labeling is accurately depicted but thenumber of extra-SCN fibers shown is less than the numberactually present, by a factor of 2:1 to 4:1.

Telencephalon. Four areas in the telencephalon wereexamined for their possible input to the SCN: the infralim-bic cortex, the septal nuclei, the substantia innominata,and the ventral subiculum. In one experiment (R140), theBDX injection site includes both infralimbic and prelimbiccortices (Fig. 7A, left). In this case, axonal labeling in theSCN is largely confined to the dorsolateral part of thenucleus, with only a few fibers present elsewhere in theSCN (Fig. 7A, right). Many labeled fibers are found in theperi-SCN region, immediately outside the SCN (Fig. 7A,right).

Fig. 6. Photomicrographs of three representative biotinylated dextran (BDX) injection sites. A:Arcuate nucleus of the hypothalamus. B: Ventral subiculum. C: Median raphe nucleus. Scale bar 5 200 µm.


Fig. 7. Camera lucida drawings illustrating the distribution ofaxonal labeling in the SCN and peri-SCN region following BDXinjections into the (A) infralimbic cortex (experiment R140), (B) thelateral septum (experiment R141), and the (C) substantia innominata

(experiment R142). For each experiment, the BDX injection site(stippled area) is illustrated on the left, and the SCN/peri-SCNterminal field is depicted on the right. For abbreviations, see list.


Fig. 8. Camera lucida drawings illustrating the distribution ofaxonal labeling in the SCN and peri-SCN region following (A) BDXinjection into the ventral subiculum (experiment R158), (B) BDXinjection into the subfornical organ (experiment R180), and (C)Phaseolus vulgaris-leucoagglutinin (PHA-L) injection into the paraven-

tricular thalamic nucleus (experiment B7). For each experiment, theinjection site (stippled area, extent of the BDX injection; black dots,labeled cells within the PHA-L injection site) is illustrated on the left,with the corresponding SCN/peri-SCN terminal field on the right. Forabbreviations, see list.


Two experiments have injection sites in the septalnuclei. The BDX injection site in experiment R141 iscentered in the ventrolateral part of the lateral septum(Fig. 7B, left). In this case, a very dense plexus is locatedimmediately lateral to the SCN in the anterior hypotha-lamic area (Fig. 7B, right). A moderately dense plexus isobserved in the dorsolateral SCN with a few scatteredfibers in the dorsomedial and ventral SCN (Fig. 7B, right).Fibers-of-passage, grouped in small bundles, are presentalong the dorsomedial border of the SCN (Fig. 7B, right).In PHA-L experiment R150, the injection site is centeredin the rostral, medial part of the lateral septum, andincludes the medial septum and the vertical limb of thediagonal band. Only a few fibers are found in the SCN inthis experiment, and these are concentrated dorsally andlaterally, similar to experiment R141.

After a BDX injection into the substantia innominata, afew varicose fibers are present in the dorsal part of theSCN (Fig. 7C). Relatively little axonal labeling is foundoutside the SCN (Fig. 7C) compared with experimentsR140 (Fig. 7A) and R141 (Fig. 7B). In BDX experimentR158, with an injection site centered in the ventral subicu-lum, a rich plexus of fibers is observed outside the SCN inthe anterior hypothalamic area (Fig. 8A). In this experi-ment, a few labeled fibers extend into the dorsal SCN; theventral SCN is devoid of labeling (Fig. 8A, right; Fig. 9A).

Diencephalon. In two experiments (R180 and R184),the BDX injection sites are located in the subfornicalorgan. Both injection sites are small and centered in thedorsal part of the central core region (experiment R180;Fig. 8B, left). The injection site in experiment R180 isslightly larger than that in experiment R184 and includespart of the outer rim where retrogradely labeled cells areobserved after SCN FG injections. In this experiment, asmall number of labeled fibers with boutons is present inthe SCN and the adjacent periventricular area (Fig. 8B,right).

In two experiments (B5 and B7) with PHA-L injectionsin the rostral, dorsal part of the paraventricular thalamus,a moderately dense plexus of PHA-L labeled fibers is foundin the SCN (Figs. 8C, 9B). This terminal field includes theentire SCN, although labeled fibers are most numerous inthe anterior and dorsal SCN (Figs. 8C, 9B). Many labeledfibers with boutons extend beyond the dorsal border of theSCN and terminate along the ventricular surface (Fig. 8C).

Several areas in the rostral hypothalamus were exam-ined for their possible input to the SCN. In experimentR192, the BDX injection site is centered immediatelybeneath the anterior commissure and slightly lateral tothe midline, and includes the median preoptic nucleus aswell as the adjacent medial preoptic area. In this experi-ment, a conspicuous plexus of labeled fibers surrounds theSCN (i.e., the peri-SCN area), with a small number offibers located within the dorsal SCN (Fig. 9C). The ventralSCN is largely devoid of labeling. In experiment R186, theBDX injection site is quite large, and includes the lateralpart of the medial preoptic nucleus and the adjacentmedial preoptic area. In this case, a dense terminal plexusis present in the peri-SCN region with relatively sparseaxonal labeling in the dorsal half of the SCN. Only a fewfibers are present in the ventral SCN, and these arelocated adjacent to the optic chiasm. In experiment R181,with a BDX injection site centered in the anterior hypotha-lamic area lateral to the SCN, the axonal labeling in the

SCN shows a distribution identical to that observed inexperiments R186 and R192.

In three experiments (R30, R76, and R79), the PHA-Linjection site is located in the rostral half of the VMH. Theaxonal labeling in the SCN is identical for all threeexperiments. In representative experiment R79, denseaxonal labeling is present outside the SCN in the anteriorhypothalamic and medial preoptic areas (Fig. 10A). Manylabeled fibers are located in the tractus infundibularis,located between the suprachiasmatic nuclei (Fig. 10A,right). Within the SCN, many labeled fibers are found inthe dorsal part of the nucleus: A few fibers are also presentin the ventral SCN along the optic chiasm (Fig. 10A, right).One BDX injection was placed in the arcuate nucleus of thehypothalamus (experiment R152; Fig. 10B, left). Likemany of the other hypothalamic afferents to the SCN, thisprojection also terminates preferentially in the dorsal SCN(Fig. 10B, right).

The projection from the posterior hypothalamic area tothe SCN is evident in four experiments with PHA-L as theanterograde tracer. In three of these cases, the injectionsites are centered in the posterior hypothalamic area(experiments R136, R153, and R154), and, in the fourthcase, the injection site is located in the dorsal hypotha-lamic area but includes a small portion of the posteriorhypothalamic area (experiment R155). The pattern ofaxonal labeling in the SCN is similar for all four experi-ments. In experiment R153, the PHA-L injection site islocated in the most ventral part of the PHA (Fig. 10C, left).In this case, labeled fibers with boutons are found in thedorsal part of the SCN (Fig. 10C, right). The injection sitein experiment R154 is somewhat more dorsal than that inexperiment R153. In this case, there is increased labelingin the dorsal SCN with a few fibers also present in theventral SCN. In experiment R136, the injection site isquite small and is located between the third ventricle andthe fields of Forel. In this case, a few scattered fibers withboutons are present in the rostral and dorsal SCN. Inexperiment R155, axonal labeling is sparse in the SCN,with only a few fibers in the dorsomedial subdivision.

In three experiments (IGL17, IGL19, and IGL28), thePHA-L injection site is centered in the IGL. The pattern ofaxonal labeling in the SCN is identical for all threeexperiments. In representative experiment IGL17, theinjection site is small and centered in the IGL at itsmid-rostrocaudal extent (Fig. 11A, left). This injection siteincludes a few cells in the dorsal lateral geniculate nucleusand the medial parvocellular part of the ventral lateralgeniculate nucleus. Axonal labeling is quite dense inventrolateral portions of the SCN (Figs. 9D, 11A, right).Some fibers terminate lateral and dorsal to the SCN (Fig.11A, right).

Two animals received BDX injections into the pretec-tum. In experiment R179, with an injection site centeredin the lateral part of the medial pretectal area, a largenumber of labeled fibers is present beneath the SCN in thesupraoptic commissures (Fig. 11B). Labeled fibers withboutons are particularly prominent in the most ventrolat-eral part of the SCN and in the area immediately lateral tothe SCN (Fig. 11B, right). In experiment R178, the injec-tion site is centered in the posterior pretectal nucleus butincludes the olivary pretectal nucleus at more rostrallevels (Fig 11C, left). In this case, a few fibers are presentin the rostral part of the ventrolateral SCN, adjacent to theoptic chiasm. With subsequent sections, the axonal label-


Fig. 9. Photomicrographs of axonal labeling in the SCN (dorsome-dial, dm; ventrolateral, vl) in four different anterograde experiments.A: In experiment R158, with a BDX injection site in the ventralsubiculum, a few labeled fibers extend into the dorsolateral part of theSCN from the adjacent anterior hypothalamic area. B: A cluster ofPHA-L labeled fibers in the SCN after an injection into the paraven-

tricular thalamic nucleus (B7). C: After a BDX injection into themedian preoptic nucleus (R192), many labeled fibers are present in thedorsal half of the SCN with numerous labeled fibers in the peri-SCNarea. D: A dense concentration of axonal labeling in the ventrolateralpart of the SCN after a PHA-L injection into the IGL (IGL17). Forabbreviations, see list. Scale bar 5 100 µm.


Fig. 10. Camera lucida drawings illustrating the distribution ofaxonal labeling in the SCN and the peri-SCN region following (A)PHA-L injection into the VMH (R79), (B) BDX injection into thearcuate nucleus (R152), and (C) PHA-L injection into the posteriorhypothalamic area (R153). For each experiment, the injection site

(stippled area, extent of the BDX injection; black dots, labeled cellswithin the PHA-L injection site) is illustrated on the left, with thecorresponding SCN/peri-SCN terminal field on the right. For abbrevia-tions, see list.


Fig. 11. Camera lucida drawings illustrating the distribution ofaxonal labeling in the SCN and peri-SCN region following (A) PHA-Linjection into the IGL (IGL17), (B) BDX injection into the medialpretectal area (R179), and (C) BDX injection into the olivary/posterior

pretectal nuclei (R178). For each experiment, the injection site (stippledarea, extent of the BDX injection; black dots, labeled cells within thePHA-L injection site) is illustrated on the left, with the correspondingSCN/peri-SCN terminal field on the right. For abbreviations, see list.


ing is found in the center of the SCN and then, at morecaudal levels, within the dorsolateral SCN (Fig. 11C,right). Labeled fibers with boutons are conspicuous justlateral to the SCN in the peri-SCN region (Fig. 11C, right).

Brainstem. Six animals received BDX injections intothe dorsal and median raphe nuclei. Two injections (experi-ments R143 and R187) are located in the median raphe.One injection site (experiment R193) is centered in theventromedial cell group of the midbrain dorsal raphe. Theother three injection sites (experiments R199, R200, andR203) involve the lateral cell group of the midbrain dorsalraphe (for dorsal raphe cell groups, see Tork, 1985). Themost dense fiber labeling in the SCN is observed afterinjections into the median raphe. In experiment R143, theinjection site is centered in the caudal, ventral part of themedian raphe nucleus and includes both the midbrain andpontine median raphe nuclei (Fig. 12A, left). In this case,many fine, lightly stained fibers are found in the ventralSCN (Fig. 12A, right). A few, darkly stained, coarse fiberswith boutons are present in the dorsolateral SCN; thistype of fiber is more numerous outside the SCN (Fig. 12A,right). A similar plexus of fine fibers is present in theventral SCN of experiment R187, with an injection sitecentered in the midbrain paramedian raphe nucleus (Fig.13A).

Axonal labeling is scarce in the SCN after injections intothe dorsal raphe nucleus. In experiment R193, with asmall BDX injection site in the rostral ventromedial cellgroup of the dorsal raphe, two or three fibers are present inthe SCN. In contrast, many labeled fibers with boutons arefound in the supraoptic commissures and in the tractusinfundibularis. After a discrete BDX injection into thelateral cell group of the midbrain dorsal raphe (experimentR199), a moderately dense plexus of coarse, darkly stainedfibers is observed in the anterior hypothalamic area imme-diately outside the SCN (Fig. 13B). A few fibers are locatedwithin the SCN (Fig. 13B). Ventral to the SCN, manyfine-caliber, lightly stained fibers are observed travellingin the supraoptic commissures (Fig. 13B). The axonallabeling in experiment R203, which has a large injectionsite that includes both the central gray and the lateral cellgroup of the dorsal raphe, shows a distribution similar tothat observed in experiment R199. In this case, smallnumbers of fibers are scattered in the SCN, and manyfibers with boutons are located dorsal and lateral to theSCN in the peri-SCN region. In experiment R200, the BDXinjection site is centered in the ventral lateral central gray,with some involvement of the lateral cell group of thedorsal raphe nucleus. In this case, many varicose fibers arefound in the dorsal part of the supraoptic commissures,with a few fine-caliber fibers located within the SCN.

In one experiment (R156), a BDX injection was placed inthe central lateral subnucleus of the parabrachial nucleus(Fig. 12B, left). A moderately dense plexus of fibers isobserved in the peri-SCN area and in the tractus infundibu-laris, with small numbers of labeled fibers also locatedwithin the SCN (Fig. 12B, right). One animal received asmall, discrete BDX injection into the locus coeruleus(R146). In this case, a few scattered fibers are present inthe SCN and in the underlying supraoptic commissures.

Several brain areas, which are labeled in our FG experi-ments, project heavily to areas immediately adjacent tothe SCN, with little or no input to the SCN itself. After a

BDX injection into the lateral half of the superior collicu-lus, with involvement of both the superficial gray and theoptic nerve layers (experiment R84), a large number offibers is present in the ventral portion of the supraopticcommissures at a mid-rostrocaudal SCN level (Fig. 14A). Afew labeled fibers extend into the ventral subdivision of theSCN (Fig. 14A). Given the wide expanse of the superiorcolliculus, and the diffuse distribution of retrogradelylabeled cells in this nucleus after SCN FG injections, theprojection from the superior colliculus to the SCN may bemore substantial than indicated here. In experiment R52,with an injection centered in the parabigeminal nucleus,numerous fibers are observed in the supraoptic commis-sures, decussating at a rostral SCN level. At more caudallevels, these fibers are closely adjacent to, but do notappear to terminate in, the ventral SCN. In case R144, theBDX injection site is located in the most caudal, medialportion of the pedunculopontine nucleus and includes theadjacent laterodorsal tegmental nucleus (Fig. 12C, left). Inthis case, many labeled fibers are observed in the dorsalportion of the supraoptic commissures, decussating at amidrostrocaudal SCN level (Figs. 12C, right, 14B). A fewlabeled fibers with boutons extend into the SCN from theanterior hypothalamic area.

DISCUSSION

Two principal findings emerge from our anatomicalstudies of the SCN afferent connections. First, the SCNreceives afferent input from six major areas: the retina, thelimbic system, the hypothalamus, the raphe nuclei, theparaventricular thalamus, and the extraretinal visualsystem (e.g., IGL, pretectum). Second, the afferent inputsare organized topographically within the SCN and adja-cent peri-SCN region (Fig. 15).

Retinal input to the SCN

Previous studies have shown that the rat retinohypotha-lamic tract terminates predominantly within the ventralSCN (Moore and Lenn, 1972; Johnson et al., 1988b; Levineet al., 1991; Speh and Moore, 1992, 1993); thus, we did notreexamine this projection in the present study. For com-pleteness, we have included the SCN retinal terminal field(Fig. 15), which was illustrated in Figure 1D of Speh andMoore (1993). The role of the retinal input in photicentrainment of the circadian pacemaker has been exten-sively reviewed (Meijer, 1991; Van den Pol and Dudek,1993; Morin, 1994; Moore, 1995; Moore et al., 1995).

Raphe inputs to the SCN

The SCN receives one of the most dense serotonergicinnervations in the hypothalamus (Halliday et al., 1995).Serotonin-containing fibers in the SCN are concentrated inthe ventral subdivision (Ueda et al., 1983; Van den Pol andTsujimoto, 1985). Two early autoradiographic studies iden-tified the midbrain raphe nuclei as the most likely sourcefor these fibers (Azmitia and Segal, 1978; Moore et al.,1978). Moore et al. observed dense labeling in the ventralportion of the SCN after a [3H]proline injection into themidbrain raphe nuclei. For evidence of the serotonergicnature of this projection, they observed no labeling in theSCN of an experimental animal that received the seroto-nin neurotoxin, 5,6-dihydroxytryptamine, prior to the


Fig. 12. Camera lucida drawings illustrating the distribution ofaxonal labeling in the SCN and peri-SCN region following BDXinjections into (A) the median raphe nuclei (R143), (B) the parabra-chial nucleus (R156), and (C) the pedunculopontine and laterodorsal

tegmental nuclei (R144). For each experiment, the BDX injection site(stippled area) is illustrated on the left, with the correspondingSCN/peri-SCN terminal field on the right. For abbreviations, see list.


[3H]proline injection. In a similar study, Azmitia andSegal, concluded after [3H]proline injections restricted toeither the dorsal or the median raphe, that the SCNreceives input from both raphe nuclei. In support of thisfinding, two retrograde tracing studies have reportedlabeling in both the dorsal and the median raphe afterSCN injections (Pickard, 1982; Bina et al., 1993). In ourFG experiments, we observe retrogradely labeled neuronsin three raphe nuclei: the midbrain dorsal raphe nucleus,the midbrain median raphe nucleus, and the pontinemedian raphe nucleus. Labeled neurons are distributedequally across the dorsal and median raphe nuclei. Two tothree times more neurons are observed in the midbrainmedian raphe than in the pontine median raphe. Withinthe dorsal raphe nucleus, only a few labeled cells arepresent in the central (i.e., dorsomedian and ventromedial)cell groups; the majority of cells are located bilaterally inthe lateral cell groups (for a description of these cellgroups, see Tork, 1985). This pattern of retrograde labelingis similar to the distribution of serotonergic cells projectingto the paraventricular hypothalamic nucleus (Sawchenkoet al., 1983).

In agreement with recent findings in the hamster (Meyer-Bernstein and Morin, 1996), our anterograde tracing re-sults suggest that the median raphe nuclei are the majorsource of serotonergic innervation of the rat SCN. In twoBDX experiments with median raphe injections, we ob-serve many fine-caliber labeled axons concentrated in theventral subdivision of the SCN (Figs. 12A, 13A). TheseBDX-labeled fibers closely resemble serotonin-immunore-active fibers in terms of their size, morphology, and

distribution within the SCN (Ueda et al., 1983). In con-trast, the SCN shows a lack of axonal labeling after a smallBDX injection centrally placed in the dorsal raphe nucleus(specifically located in the ventromedial cell group). Inagreement with this finding, Vertes (1991) noted an ab-sence of labeling in the SCN after PHA-L injectionscentered in the dorsal raphe nucleus. Evidence for amedian raphe serotonergic input to the SCN can be foundin an early study by Van de Kar and Lorens (1979), whoshowed that the serotonin content of the SCN significantlydecreases after discrete lesions of the midbrain medianraphe but not after similar-sized lesions in the dorsalraphe.

The lateral cell group of the dorsal raphe nucleus maynot have been included in the PHA-L injections of Vertes(1991) or in the lesions of Van de Kar and Lorens (1979).Our results suggest that this part of the dorsal raphe mayprovide an additional, although minor, serotonergic inputto the SCN. In the present study, we observed a densecollection of fibers surrounding the SCN, but only a smallnumber of fibers within the SCN, after BDX injectionscentered in the lateral cell group of the dorsal raphenucleus. The labeled fibers in the SCN showed variablemorphology and no preferential distribution, unlike thefibers from the median raphe nuclei, suggesting that theymay arise from both serotonergic and nonserotonergicneurons in the dorsal raphe. In these same experiments,many fine-caliber, serotonin-like fibers are present in thesupraoptic commissures immediately beneath the SCN.These fibers continue caudally into the ventral lateralgeniculate nucleus. Double-labeling studies have previ-

Fig. 13. Photomicrographs of axonal labeling in the SCN following BDX injections into (A) the medianraphe nucleus (R187) and (B) the lateral cell group of the dorsal raphe nucleus (R203). In A, note the denseconcentration of fine-caliber fibers in the ventral part of the SCN. In B, note the relative lack of SCNaxonal labeling. Scale bar 5 100 µm.


ously described serotonergic inputs from the lateral cellgroup to the retina, superior colliculus, and lateral genicu-late nucleus (Villar et al., 1987, 1988). It is possible thatsome of our FG injection sites, particularly those withdamage to the underlying supraoptic commissures, la-beled fibers-of-passage travelling from the dorsal raphelateral cell group to the ventral lateral geniculate nucleusand the superior colliculus. Thus, only a small portion ofthe labeled cells in the dorsal raphe lateral cell group inour retrograde experiments may actually project to the SCN.

IGL/pretectal inputs to the SCN

In addition to a direct photic input from the retina, theSCN receives indirect photic input from retinorecipientareas, such as the IGL and the pretectum. The IGL sends adense projection to the SCN (Mikkelsen, 1990; presentstudy). This projection terminates in the ventrolateralSCN in a pattern that is identical to that reported forneuropeptide Y-immunoreactivity (Card and Moore, 1988).Previous studies have shown that neuropeptide Y-immuno-reactive neurons in the IGL project to the SCN (Card andMoore, 1982, 1989; Harrington et al., 1985, 1987; Morin etal., 1992; Janik and Mrosovsky, 1994). Infusion of neuropep-tide Y into the SCN of a free-running animal elicits aphase-dependent phase shift in the animal’s circadianrhythm of locomotor activity (Albers and Ferris, 1984;Huhman and Albers, 1994). The phase-response curve(PRC; magnitude of phase shift plotted against circadiantime of administration) of neuropeptide Y is very similar tothe PRC obtained with nonphotic stimuli that induce

locomotor activity (Reebs et al., 1989; Van Reeth andTurek, 1989), suggesting that the geniculohypothalamictract may mediate nonphotic entrainment of the circadianpacemaker. Consistent with this possibility, bilateral le-sions of the IGL block the locomotor activity-induced phaseshifts that occur in response to benzodiazepine administra-tion (Johnson et al., 1988a; Biello et al., 1991) or exposureto a novel running wheel (Janik and Mrosovsky, 1994;Wickland and Turek, 1994). Further discussion of the roleof the IGL in circadian timing may be found in Medanicand Gillette (1993), Moore and Card (1994), and Morin(1994).

Our description of the pretectal projection to the SCN isin general agreement with the recent report of Mikkelsenand Vrang (1994). In both studies, retrogradely labeledcells are present in the olivary pretectal nucleus, themedial pretectal area, and the posterior pretectal nucleusfollowing tracer injections into the SCN. As an extension totheir findings, that the pattern of retrograde labeling inthe pretectum varies with the SCN injection site. Neuronsin the olivary pretectal nucleus are retrogradely labeled ineach of our FG experiments. The posterior pretectal nucleusshows very little retrograde labeling, except in cases withinjection site involvement of the anterior hypothalamicarea (e.g., experiment 93-87). Likewise, retrogradely la-beled cells in the medial pretectal area are most numerousin experiments with an injection site that includes eitherthe supraoptic commissures or the anterior hypothalamicarea. This topography is also apparent in our anterogradeexperiments. A BDX injection into the medial pretectal

Fig. 14. Photomicrographs of axonal labeling in the supraoptic commissures ventral to the SCN afterBDX injections into (A) the superior colliculus (R84) and (B) the caudal, medial part of the pedunculopon-tine nucleus (R144). In A, note the small number of labeled fibers present in the ventral SCN. Scale bar 5100 µm.


area produces extensive labeling in the supraoptic commis-sures, dense labeling in the area lateral to the SCN, anddiffuse labeling within the confines of the SCN. In experi-ment R179, with an injection site that includes bothposterior and olivary pretectal nuclei, axonal labeling isconcentrated in the area lateral to the SCN with only asmall number of labeled fibers within the SCN. The

pattern of SCN labeling in this experiment (R179) is verysimilar to the distribution depicted in Figure 3 of Mik-kelsen and Vrang (1994). However, our delineation of theSCN appears to be more conservative than these investiga-tors, as we locate the most prominent cluster of fibers inthe anterior hypothalamic area, immediately lateral to theSCN, rather than in the SCN itself.

Fig. 15. Summary schematic of the six major terminal fields withinthe SCN and adjacent peri-SCN region. The retinal terminal field isbased on Figure 1D of Speh and Moore (1993). The median raphedistribution is derived from Figure 13A. The IGL and pretectaldistribution was created by superimposing the terminal fields of theIGL (Fig. 11A), the medial pretectal area (Fig. 11B), and the olivary/posterior pretectal nuclei (Fig. 11C). The thalamic terminal field is

based on Figure 8C. The hypothalamic distribution was created bysuperimposing the terminal fields of the medial preoptic area (notshown), the ventromedial nucleus (Fig. 10A), the arcuate nucleus (Fig.10B), and the posterior hypothalamic area (Fig. 10C). The limbicterminal field represents the combined terminal fields of the infralim-bic cortex (Fig. 7A), the lateral septum (Fig. 7B), and the ventralsubiculum (Fig. 7C).

527M.M. MOGA AND R.Y. MOORE

Thalamic input to the SCN

The paraventricular thalamus sends a moderately denseprojection to the SCN, innervating both dorsomedial andventrolateral subdivisions (Fig. 15; Moga et al., 1995). Thisprojection may be a source of excitatory input to SCNneurons. Excitatory amino acids are the principal neuro-transmitters in thalamic relay neurons (Steriade andLlinas, 1988; Kharazia and Weinberg, 1994). Anatomicalevidence for excitatory neurotransmission in thalamicoutput neurons is based in part on the results of [3H]D-aspartate tracing studies (Ottersen et al., 1983; Christie etal., 1987; Fuller et al., 1987; Carnes et al., 1990). [3H]D-aspartate is a false neurotransmitter that is taken up bythe high-affinity glutamate/aspartate uptake system andretrogradely transported to the cell somata (Cuenod andStreit, 1983; Fuller et al., 1987). After [3H]D-aspartateinjections into the striatum and ventral pallidum, manyretrogradely labeled neurons are present in the midlinethalamus, including the paraventricular thalamic nucleus;these labeled neurons are presumably glutamatergic and/oraspartatergic (Christie et al., 1987; Fuller et al., 1987).Neurons in the paraventricular thalamus are retrogradelylabeled following [3H]D-aspartate injections into the SCNof the rat (Moga and Moore, 1996) and the hamster (DeVries and Lakke, 1995). We speculate that some of thenonretinal glutamatergic terminals previously detected inthe SCN at the ultrastructural level (Castel et al., 1993; DeVries et al., 1993) may originate from paraventricularthalamic neurons.

Lesions of the paraventricular thalamus in the hamsterproduce no change in the free-running circadian rhythm oflocomotor activity or in the animal’s ability to entrain tothe light-dark cycle (Ebling et al., 1992), suggesting thatthis nucleus is not crucial for circadian rhythm generationor for photic entrainment of circadian rhythms. Alterna-tively, the paraventricular thalamic nucleus may be in-volved in nonphotic entrainment of the circadian pace-maker. By using the expression of the immediate earlygene c-fos as a metabolic marker, investigators havedemonstrated that paraventricular thalamic neurons areactivated by a variety of nonphotic stimuli, includingimmobilization stress (Chastrette et al., 1991; Senba et al.,1993; Cullinan et al., 1995), noxious chemical and mechani-cal stimuli (Senba et al., 1993), peripheral administrationof peptide YY (Bonaz et al., 1993), intracerebroventricularadministration of nerve growth factor (Peng et al., 1993),stimulation of the femoral nerve (McKitrick and Calaresu,1993), and an appetitive conditioned stimulus (Brown etal., 1992). A series of double-labeling experiments, combin-ing injections of retrograde tracer into the SCN with Fosimmunohistochemistry, may be useful in identifying thetype of information being relayed from the paraventricularthalamus to the SCN.

Hypothalamic inputs to the SCN

The hypothalamic input to the SCN is widespread inorigin, arising from at least 11 different cell groups,including the medial preoptic and median preoptic nuclei;the anteroventral periventricular, ventromedial, dorsome-dial, tuberomammillary, and arcuate hypothalamic nuclei;the subparaventricular zone; and the medial preoptic,lateral hypothalamic, and posterior hypothalamic areas.In general, hypothalamic cell groups project to the dorsalsubdivision of the SCN, largely avoiding the VIP cell

population in the ventral SCN (Fig. 15). In individualexperiments with tracer injections in the hypothalamus,the axonal labeling in the dorsal part of the SCN is quitesparse, suggesting the hypothalamic inputs to be of minorimportance in circadian function. However, if we considerthe total of each of these inputs, we are led to theconclusion that the hypothalamus as a whole provides asubstantial input to the SCN, particularly to the dorsalSCN. Furthermore, each of the hypothalamic afferent cellpopulations projects to the peri-SCN region (Figs. 9C, 10,15). Some SCN dendrites extend in this region (Van denPol, 1980), increasing the possibility for synaptic contactbetween the hypothalamus and the SCN.

The preoptic projection to the SCN originates from cellsin the medial preoptic area, the median preoptic nucleus,the medial preoptic nucleus, and the anteroventral periven-tricular nucleus (Pickard, 1982; Simerly and Swanson,1988; Bina et al., 1993; present study). After BDX injec-tions into the preoptic hypothalamus, we observe a denseplexus of fibers in the peri-SCN region (Fig. 9C). Somelabeled fibers extend into the SCN to terminate within thedorsal part of the nucleus (Fig. 9C). Simerly and Swansonfound an identical pattern of labeling after PHA-L injec-tions into the medial preoptic nucleus.

Gonadotropin-releasing hormone (GnRH) may be a neu-romodulator within the preoptic projection to the SCN.The distribution of GnRH-immunoreactive fibers in theSCN (Van der Beek et al., 1997) shows extensive overlapwith the preoptic terminal field. Furthermore, GnRH-immunoreactive cells are present in areas of the preoptichypothalamus, such as the vascular organ of the laminaterminalis and the medial preoptic area, that containSCN-projecting cells (Kawano and Daikoku, 1981; Witkinet al., 1982; Van der Beek, 1996). A double-labeling study isneeded to substantiate this neuropeptide projection.

The projection from the VMH to the SCN was describedin several early anatomical studies using either axonaldegeneration or retrograde tracing (Szentagothai et al.,1968; Moore et al., 1979; Zaborszky and Makara, 1979;Pickard, 1982). Anterograde tracing evidence for thisprojection has been less forthcoming. In two early autora-diographic studies of the VMH efferent projections, adense concentration of silver grains was observed surround-ing the SCN with very few grains located within thenucleus, suggesting a lack of innervation by the VMH(Saper et al., 1977; Krieger et al., 1979). In a recent PHA-Lstudy, Canteras et al. (1994) depict a few labeled axons inthe SCN following injections into the VMH (see their Figs.3 and 5), but they do not describe this relatively minorVMH projection. Like many of the SCN hypothalamicafferents, the VMH preferentially innervates the dorsalsubdivision (present study). In turn, the SCN sends areciprocal projection to the VMH (Watts and Swanson,1987; Watts et al., 1987; Morin et al., 1994).

The function of the VMH input to the SCN is problem-atic. VMH lesions significantly dampen the circadianrhythms of feeding and corticosterone release (Bernardis,1973; Bellinger et al., 1976; Danguir and Nicolaidis, 1978;Egawa et al., 1993). Interpretation of these lesion effects iscomplicated by lesion site involvement of both the SCNterminal field and the SCN afferent cell population locatedwithin the nucleus (Pickard, 1982; Watts and Swanson,1987; Watts et al., 1987; Morin et al., 1994; present study).Chronic stimulation of the VMH with a hydropolymer gel,


which supposedly leaves SCN axons in the VMH intact,also attentuates the expression of the circadian activityrhythm (Mitsushima et al., 1994). However, knife cutsaround the gel-stimulated VMH indicate that this effect ismediated through a dorsal projection of the VMH, possiblyto the paraventricular thalamic nucleus, and not througha direct input to the SCN.

Our results show that the arcuate nucleus sends a smallprojection to the SCN. In an early electrophysiologicalstudy, Makara and Hodacs (1975) observed an antidromicactivation of arcuate neurons following electrical stimula-tion of the suprachiasmatic/preoptic region, indicating adirect pathway from the arcuate nucleus to the SCNand/or preoptic area. More conclusive evidence for anarcuate-SCN pathway was provided by Van den Pol andTsujimoto (1985), who reported adrenocorticotropin(ACTH)-immunoreactive fibers in the rat SCN. Becausethe arcuate nucleus is the primary source of ACTH inner-vation in the rat forebrain (Chronwall, 1985; Joseph andMichael, 1988), the ACTH-immunoreactive fibers in theSCN are most likely derived from the arcuate nucleus.

The dorsomedial hypothalamic nucleus sends a denseprojection to the peri-SCN region with minor input to thedorsal SCN (Thompson et al., 1996). In agreement withthis finding, we observe retrogradely labeled neurons inthe dorsomedial nucleus after injections of FG into theSCN and peri-SCN region. The neuropeptide galanin maybe present within this pathway. The dorsomedial nucleuscontains many galanin-immunoreactive neurons, and gala-nin-immunoreactive fibers are numerous within the peri-SCN and dorsal SCN (Merchenthaler et al., 1993). In adouble-labeling study, Levin et al. (1987) found that thedorsomedial nucleus is a major source of galaninergicinput to the paraventricular hypothalamic nucleus. Asimilar double-labeling study is needed to determinewhether the SCN and peri-SCN also receive galaninergicinput from the dorsomedial nucleus.

Contrary to the other hypothalamic afferents, neuronsin the subparaventricular zone and tuberomammillarynucleus may innervate both dorsal and ventral SCNsubdivisions. Swanson and colleagues have reported adiffuse pattern of innervation in the SCN following PHA-Linjections into the subparaventricular zone (Swanson,1987; Watts et al., 1987). The subparaventricular zone islocated immediately ventral to the paraventricular nucleusand medial to the anterior hypothalamic nucleus. Thiszone is a major terminal field for SCN efferent projections(Watts, 1991) as well as a source of afferent input to theSCN (Pickard, 1982; Watts et al., 1987; Morin et al., 1994;present study).

Evidence suggests that the tuberomammillary nucleusprojects to both SCN subdivisions. A few magnocellularneurons in the ventral subgroup of the tuberomammillarynucleus (for subgroups, see Ericson et al., 1987; Inagaki etal., 1990) are retrogradely labeled in each of our FGexperiments. The tuberomammillary nucleus is the soleneuronal source of histamine in the mammalian brain (forreview, see Schwartz et al., 1991). Histaminergic termi-nals are numerous in the rat SCN (Inagaki et al., 1988;Panula et al., 1989); however, the subnuclear distributionof these fibers has not been described. In a photomicro-graph in Panula et al. (1989), the histaminergic terminalsappear to be distributed evenly across both SCN subdivi-sions. Thus, the tuberomammillary and subparaventricu-

lar inputs may be unique among SCN hypothalamicafferents in their subnuclear distribution.

The histaminergic input may be of some importance incircadian timing. In free-running rats under constantdarkness, histamine elicits a phase delay in the circadianrhythms of locomotor activity and drinking behavior whenit is administered during the early part of the animal’sactive period (Itowi et al., 1990). A similar phase delay isobserved with light pulses delivered during early subjec-tive night (Daan and Pittendrigh, 1976). However, unlikephotic stimulation of the circadian pacemaker, histaminedoes not cause a permanent phase advance during latesubjective night (Itowi et al., 1990).

Limbic inputs to the SCN

The SCN receives input from three areas in the limbictelencephalon (for definition of the limbic region, seeSwanson, 1983), namely from the infralimbic cortex, thelateral septal nucleus, and the ventral subiculum. Each ofthese areas terminates primarily in the dorsal SCN subdi-vision (Fig. 15). Like the hypothalamic afferents, thelimbic afferents also project heavily to the peri-SCN re-gion, suggesting this region as a possible locus for interac-tions between the limbic system and the circadian pace-maker.

A projection from the infralimbic cortex to the SCN waspreviously described by Hurley et al. (1991). After injec-tions of PHA-L into the infralimbic cortex, they observed apreferential distribution of labeled fibers in the dorsalSCN, in agreement with our BDX results. Functionalstudies have focused on the role of the infralimbic cortex invisceral motor function (for review, see Neafsey, 1990). In arecent review of the olfactory system, Shipley et al. (1995)suggested that the infralimbic cortex, based on its inputfrom olfactory areas, may activate visceral motor areas inresponse to olfactory stimuli. In a related role, the infralim-bic cortex may serve as a link between the olfactory systemand the circadian timing system, providing an anatomicalbasis for the effects of olfactory bulbectomy on circadiantiming (Possidente et al., 1990; Pieper and Lobocki, 1991).Olfactory information may also reach the SCN throughfibers-of-passage in the supraoptic commissures; some ofthese fibers originate in the anterior cortical amygdala andthe medial part of the olfactory tubercle, both of whichreceive direct input from the olfactory bulb (Krettek andPrice, 1977).

A septal projection to the SCN was previously noted inthe rat (Garris, 1979; Staiger and Wouterlood, 1990) andthe guinea pig (Staiger and Nurnberger, 1991). This inputoriginates from small, oval-shaped neurons in the ventralsubdivision of the lateral septal nucleus and terminatespredominantly in the dorsal subdivision of the SCN (pre-sent study). g-Aminobutyric acid (GABA)-immunoreactiveneurons in the lateral septal nucleus have a similarmorphology and distribution (Onteniente et al., 1986),suggesting that some of the SCN-projecting neurons maybe GABAergic. Previous studies have shown that GABAer-gic neurons in the lateral septal nucleus project to theparaventricular hypothalamic nucleus, the anterior hypo-thalamic nucleus, and the lateral hypothalamic area (Jakaband Leranth, 1992; Roland and Sawchenko, 1993). Alterna-tively, an excitatory neurotransmitter, such as glutamate,may be present within the septal projection to the SCN. Insupport of this possibility, we recently observed retro-gradely labeled neurons in the lateral septal nucleus


following [3H]D-aspartate injections into the SCN (Mogaand Moore, 1996).

Retrograde tracing studies previously identified theventral subiculum as a source of neural input to the SCN(Meibach and Siegel, 1977; Pickard, 1982). This projectionarises largely from neurons in the distal part of the ventralsubiculum, although a few cells in the proximal part alsocontribute to this pathway (present study). After a BDXinjection into the distal part of the ventral subiculum, weobserved a small number of axonal fibers in the dorsalSCN and an absence of labeling in the ventral SCN. With asimilarly placed injection site, but with PHA-L as theanterograde tracer, Canteras and Swanson (1992) ob-served a small number of terminal boutons in the SCN; theprecise location of these terminals was not noted. Incontrast, Oldfield and Silverman (1985) observed axonallabeling throughout the SCN, but predominantly withinthe ventral subdivision, after HRP injections into theproximal part of the ventral subiculum. These findingssuggest that the pathway from the ventral subiculum tothe SCN may be topographically organized. Previousstudies have shown that the efferent cell populations of theventral subiculum are loosely organized along the medio-lateral or septotemporal axis (Meibach and Siegel, 1977;Witter and Groenewegen, 1990; Namura et al., 1994).Neurons in the proximal part of the ventral subiculumsend dense inputs to the entorhinal cortex, nucleus accum-bens, and dorsal subdivision of the lateral septum, whereasneurons in the distal part project heavily to the VMH,subparaventricular zone, ventral subdivision of the lateralseptum, and paraventricular thalamic nucleus (Witter andGroenewegen, 1990; Canteras and Swanson, 1992). Addi-tional experiments with injections of PHA-L or BDX intothe proximal part of the ventral subiculum are needed toconfirm the earlier HRP findings of Oldfield and Silverman(1985) and to determine whether the ventral SCN receivesinput from a subset of subicular neurons.

Other inputs to the SCN

A pathway from the subfornical organ to the SCN waspreviously demonstrated with the anterograde tracersPHA-L and HRP (Lind et al., 1982; Swanson and Lind,1986). After FG injections into the SCN, we consistentlyobserve retrogradely labeled cells along the outer marginof the subfornical organ. Angiotensin II-immunoreactivecells are similarly located in the subfornical organ, suggest-ing that this projection may be a source of angiotensin IIinnervation of the SCN (Lind et al., 1985). The SCNcontains a high density of angiotensin II receptors (Bunne-man et al., 1992; Song et al., 1992). Recently, Tang and Pan(1995) observed changes in the firing rates of SCN neuronsin vitro after picomolar applications of angiotensin II.Based on these findings, we speculate that angiotensin IImay be of some functional importance in the modulation ofSCN circadian timing.

The cholinergic inputs to the SCN were recently studiedby Bina et al. (1993). After injections of FG into the SCN,they observed double-labeled choline acetyltransferase-immunoreactive cells in the substantia innominata, themagnocellular preoptic nucleus, the diagonal band, themedial septum, the pedunculopontine nucleus, the lat-erodorsal tegmental nucleus, and the parabigeminalnucleus. To substantiate their retrograde findings, Bina etal. injected the anterograde tracer biocytin into the pedun-culopontine, laterodorsal tegmental, and parabigeminal

nuclei. In each anterograde experiment, they found la-beled fibers scattered in the SCN. Thus, the sparse cholin-ergic innervation of the SCN may arise, in part, fromneurons in the pedunculopontine, laterodorsal tegmental,and parabigeminal nuclei. In the present study, we observea small number of labeled fibers in the SCN after BDXinjections into both the pedunculopontine/laterodorsal teg-mental nuclei and the substantia innominata, indicatingthe substantia innominata as another possible source ofcholinergic input to the SCN.

In summary, the SCN afferent connections are topo-graphically organized. Three major patterns of termina-tion are found within the SCN, corresponding to thephotic/raphe, limbic/hypothalamic, and thalamic/hypotha-lamic neural inputs. Areas that receive photic input, suchas the retina, the IGL, and the pretectal area, denselyinnervate the ventral SCN subdivision but provide onlyminor innervation of the dorsal subdivision. Similarly, themedian raphe projection to the SCN terminates denselywithin the ventral subdivision and sparsely within thedorsal subdivision. A complementary pattern of axonallabeling, with labeled fibers concentrated in the dorsalSCN, is observed after anterograde tracer injections intothe hypothalamus and into limbic areas, such as theventral subiculum and infralimbic cortex. A third, lesscommon pattern of labeling, exemplified by the paraven-tricular thalamic afferents, consists of diffuse axonal label-ing throughout the SCN. These hodological differencesmay reflect a functional heterogeneity within the SCN.

ACKNOWLEDGMENTS

We thank Joan Speh for the use of her SCN FG materialand for her expert technical advice, Roger Weis for the useof his IGL PHA-L material, and Nadine Suhan for photo-graphic assistance.

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Organization of neural inputs to the suprachiasmatic nucleus in the rat