Raphe projections to the locus coeruleus in the rat

Afferent projections to the locus coeruleus from the various raphe nuclei, particularly of the midbrain (nuclei raphe dorsalis and medianus) and pons (nuclei raphe pontis and magnus), have been studied in the rat by retrograde transport methods using horseradish peroxidase (HRP). The locus coeruleus, in both its dorsomedial and ventrolateral divisions, and in its various anterior-posterior components, were injected with 0.05 μl of horseradish peroxidase following which various structures of the brainstem, particularly the raphe nuclei, were examined for HRP reactive cells. It was found that injections in most components of the locus coeruleus were associated with HRP positive cells in varying degrees of density in the nuclei raphe dorsalis, medianus, pontis, and magnus, with considerably sparser labelling in the anterior aspects of the medullary raphe nuclei pallidus and obscurus. Labelled cells were also seen in the nuclei of the solitary tract, contralateral locus coeruleus, lateral reticular areas of the pons and midbrain, nuclei pontis oralis and caudalis, vestibular nuclei, mesencephalic nucleus of the trigeminal nerve, fastigial nuclei of cerebellum and medial parabrachial nuclei. These data, showing widespread innervation of the locus coeruleus from all raphe nuclei, as well as many other brainstem areas, in the rat support the general view of heavy innervation of the locus coeruleus from both extra-raphe and raphe nulcei. These latter raphe projections, probably serotonergic in nature, provide anatomical support for the various experiments indicating considerable regulation of locus coeruleus activities, such as phasic events of REM sleep, among others, by most of the raphe nuclei. Thus, various activities of the locus coeruleus could be modulated or regulated by widespread projections from most raphe nuclei as well as several other regions of the brainstem.     

Identification of the hypothalamic site through which locus coeruleus axons decussate to reach and stimulate contralateral LH-RH neurons

Recently we reported that locus coeruleus (LC) electrical stimulation (ES) amplifies the quantity of LH released prior medial preoptic nucleus (MPN)-electrochemical stimulation (ECS). In these studies we also observed that amplification of LH release occurred only when we activated those LC neurons whose cell bodies reside contralateral to the site of MPN-ECS. Seemingly, stimulatory LC axons decussate to reach contralateral hypothalamic regions which contain LH-RH neurons. The purpose of the present study was to identify the site(s) at which such decussation(s) occur. To accomplish this we used a special knife blade to make gross transections in hypothalamic regions previously described by others as regions where LC decussations occur. Transection 1 (TI) interrupted axons coursing through the medial forebrain bundle (MFB) in the region of the posterior lateral hypothalamus. This transsection was placed ipsilateral to the side of LC-ES and it had no effect on LH patterns of concentrations which were released by MPN-ECS. However, T1 completely blocked the amplifying effects of LC stimulation on LH secretion after MPN-ECS. Transection 2 (T2) was placed in the region of the MPN, parallel to the superior sagittal sinus. The knife blade was lowered in midline to the top of the 3rd ventricle and transected all fibers which cross midline within and around the anterior commissure. LH release following MPN-ECS was not appreciably affected in these rats nor did T2 alter the amplifying effects of LC-ES on LH. However, while plasma LH peaked between 60 and 75 min and then declined towards baseline in MPN + LC-stimulated rats, it remained significantly elevated throughout the remainder of the blood collection periods (180 min) in rats receiving combined MPN + LC and T2. Transection 3 (T3) also was placed in the MPN region and differed from T2 only in that we lowered the knife to the base of sphenoid bone. Thus, T3 disrupted all fibers which cross midline in the AC region and dorsal to the optic chiiasm (dorsal supraotic decussation of LC axons). This transection did not affect LH release evoked by MPN-ECS but completely eliminated the amplifying effects of LC stimulation after MPN-ECS on LH secretion. These data indicate that stimulatory LC axons which affect LH-RH neuronal activity enter the hypothalamus ipsilateral to the site of LC-ES and then project rostrally in the MFB to the lateral preoptic area. In this region, certain stimulatory LC fibers leave the MFB and cross midline in the dorsal supraoptic decussation to reach the contralateral MPN where they affect LH-RH neuronal activity. The data obtained in rats with AC transections (T2) also suggest that inhibitory inputs from extrahypothalamic regions enter the hypothalamus and suppress or arrest LH-RH release. Disruption of these inhibitory preoptic projections results in the sustained secretion of elevated levels of LH-RH/LH following MPN + LC stimulation.     

Locus coeruleus projections to cortex: Topography, morphology and collateralization

The relationship between individual cells of origin within the nucleus locus coeruleus (LC) and the geometry and distribution of terminal fields in cortex was examined in the albino rat. Computer-assisted 3-dimensional reconstructions of the Nissl-stained LC allowed the characterization of the spatial distribution of LC cells. Similar reconstructions of the distributions of labelled cells following cortical injections of horseradish peroxidase were created. Comparisons of such reconstructions revealed that LC cells projecting to cortex were distributed throughout the compact dorsal LC. These cells were predominantly medium sized multipolar cells. Significant labelling of other morphological sub-populations of LC did not occur following cortical injections. Simultaneous injections of multiple fluorescent retrograde tracers into different cortical regions allowed the characterization of LC axon collateralization in cortex. Individual LC cells innervate functionally and cytoarchitectonically distinct cortical regions simultaneously. LC cells arborize more extensively in the anterior-to-posterior axis of cortex and exhibit relatively minimal medial-to-lateral collateralization. Individual LC cells were also shown to innervate both superficial and deep layers of a cortical region.     

Branching projections of catecholaminergic brainstem neurons to the paraventricular hypothalamic nucleus and the central nucleus of the amygdala in the rat

In this study, we have employed triple fluorescent-labelling to reveal the distribution of catecholaminergic neurons within three brainstem areas which supply branching collateral input to the central nucleus of the amygdala (CNA) and the hypothalamic paraventricular nucleus (PVN): the ventrolateral medulla (VLM), the nucleus of the solitary tract (NTS) and the locus coeruleus (LC). The catecholaminergic identity of the neurons was revealed by immunocytochemical detection of the biosynthetic enzyme, tyrosine hydroxylase. The projections were defined by injections of two retrograde tracers, rhodamine- and fluorescein-labelled latex microspheres, in the CNA and PVN, respectively. In the VLM and NTS, the greatest incidence of neurons which contained both retrograde tracers was found at the level of the area postrema. These neurons were mainly located within the confines of the A1/C1 (VLM) and A2 (NTS) catecholaminergic neuronal groups. Double-projecting neurons in the LC (A6) were distributed randomly within the nucleus. It was found that 15% in the VLM, 10% in the NTS and 5% in the LC of the retrogradely labelled cells projected via branching collaterals to the PVN and CNA. One half of these neurons in the VLM and NTS were catecholaminergic, in contrast to the LC where virtually all double-retrogradely labelled neurons revealed tyrosine hydroxylase immunoreactivity. In the other brainstem catecholaminergic cell groups (A5, A7, C3), no catecholaminergic neurons were found that supplied branching collaterals to the CNA and PVN. Our results indicate that brainstem neurons may be involved in the simultaneous transmission of autonomic-related signals to the CNA and the PVN. Catecholamines are involved in these pathways as chemical messengers. Brainstem catecholaminergic and non-catecholaminergic neurons, through collateral branching inputs may provide coordinated signalling of visceral input to rostral forebrain sites. This may lead to a synchronized response of the CNA and PVN for the maintenance of homeostasis.     

Induction and adaptation of Fos expression in the rat brain by two types of acute restraint stress

The present study was designed to examine whether both induction and adaptation of brain Fos expression during acute stress depend on the intensity and duration of stressors. For this purpose, different durations of two types of acute stress, mild (restraint) and severe (immobilization) stress, were employed. Stress-induced Fos expression was analyzed quantitatively by immunohistochemistry. Adaptation of Fos expression to the acute stressors was not apparent in the hypothalamic paraventricular nucleus (PVN) or locus coeruleus (LC) but was observed in the amygdala, hippocampus, and cerebral cortex. A higher level of Fos expression was seen in the PVN, LC, and amygdala, following severe stress than was seen following mild stress. In the hippocampus, the dentate gyrus showed reduced Fos expression in response to stressors, although both mild and severe acute stress increased Fos expression in other regions of the hippocampus. The cingulate cortex showed increased Fos expression during mild stress, whereas long-duration severe stress reduced Fos expression. In the somatosensory cortex, both stressors increased Fos expression. These results indicate that the PVN and LC are relatively resistant to adaptation to acute stress compared to other brain regions. In addition, the PVN, LC, and amygdala may play important roles in the perception of the severity of stress.     

Development and topographic organization of subicular projections to lateral septum in the rat brain

One of the main subcortical targets of hippocampal formation efferents is the lateral septum. Previous studies on the subicular projections, as a main output structure of the hippocampus, have shown a clear topographic organization of septal innervation, related to the origin of the fibres along the dorsoventral axis of the subiculum in the adult brain. In contrast, studies on the developing brain depict an extensive rearrangement of subicular projections during the prenatal period, shifting from the medial septum to the lateral septum. Our study aimed to describe the postnatal development of subicular projections to the septum. We injected anterograde tracers into the subiculum of 57 pups of different postnatal ages. Injections covered the proximodistal and dorsoventral axis of the subiculum. The age of the pups at day of tracer injection ranged from the day of birth to postnatal day 30. Analyses revealed that from the first postnatal day projections from subiculum preferentially target the lateral septum. Sparse innervation in the lateral septum was already present in the first few postnatal days, and during the following 3 weeks, the axonal distribution gradually expanded. Subicular projections to the lateral septum are topographically organized depending on the origin along the dorsoventral axis of the subiculum, in line with the adult innervation pattern. Different origins along the proximodistal axis of the subiculum are reflected in changes in the strength of septal innervation. The findings demonstrate that in case of the development of subicular projections, axonal expansion is more prominent than axonal pruning.     

Afferent projections to the self-stimulation regions of the dorsal pons, including the locus coeruleus, in the rat as demonstrated by the horseradish peroxidase technique

Afferent projections to the dorsal pons of the rat have been studied using the horseradish peroxidase (HRP) technique. HRP injections were made in each of the following regions: the vicinity of the locus coeruleus (LC); the periventncular gray, medial to the LC; the medial parabrachial region, lateral to the LC; the ventral cerebellum, dorsal to the LC; and the pontine reticular formation, ventral to the LC. Because intracranial self-stimulation (ICSS) has been obtained in these regions, the afferents have been discussed in terms of their possible contributions to that behavior. Previous ICSS studies of the dorsal pons have focussed on the LC as playing a central role. Presently identified inputs to the LC include: the dorsal raphe nucleus: the ventrolateral periaqueductal gray: the pontine reticular formation: the areas that contain the pontine and medullary noradrenergic and adrenergic cell groups: the lateral hypothalamic area: the contralateral LC: the deep cerebellar nuclei: the ventrolateral and parafascicular thalamic nuclei: and the parabrachial regions of the pons and midbrain.     

The iontophoretic application of Fluoro-Gold for the study of afferents to deep brain nuclei

A method is described for identifying the afferents to confined areas within the central nervous system using iontophoretic application of the fluorescent tracer, Fluoro-Gold (FG). Unlike other fluorescent tracers, it is possible to make focal iontophoretic injections through small-tipped micropipettes, and electrophysiological recordings from the injection pipette can be used to define structures prior to injections. Retrograde labeling with FG appears to be as sensitive as wheatgerm agglutinin-conjugated horseradish peroxidase visualized with tetramethylbenzidine. Furthermore, iontophoretically applied FG does not appear to be taken up and transported retrogradely by fibers of passage. Finally, retrograde transport of FG can be combined with immunofluorescence without: appreciable loss of sensitivity in either label.     

Telencephalic projections to the goldfish hypothalamus: An anterograde degeneration study

In this study, large areas of goldfish telencephalon were ablated including rostral nucleus preopticus periventriculare (rNPP), and degenerating axons were traced by a modified Fink and Heimer procedure. The lesioning procedure ablated large regions of area dorsalis telencephali pars medialis, centralis, and dorsolateral complex; and completely removed area ventralis telencephali pars dorsalis, ventralis, and lateralis. In addition, the supracommissural nucleus and rNPP were lesioned specifically because both nuclei have been thought to be involved in courtship behavior and endocrine control of reproduction. This investigation demonstrated extensive fiber projections from telencephalic nuclei and/or rNPP to the hypothalamus. Lesioned telencephalon and/or rNPP projected bilaterally to nucleus preopticus and the suprachiasmatic nucleus and unilaterally to the following tuberal nuclei: nucleus anterior tuberis, and the lateral hypothalamic nucleus. A much larger fiber projection to the inferior lobe nuclei was also observed with a large contralateral as well as ipsilateral input.     

Spinal projections from the nucleus locus coeruleus and nucleus subcoeruleus in the cat and monkey as demonstrated by the retrograde transport of horseradish peroxidase

The rostral pons of the cat and rhesus monkey were examined for the presence of labeled cells following injections of horseradish peroxidase (HRP) into the lumbar spinal cord. Labeled cells were found in the ipsilateral dorsolateral pontine tegmentum and in the contralateral ventrolateral pontine reticular formation. In both the cat and monkey, labeled cells were located in the nucleus locus coeruleus, nucleus subcoeruleus, in or near the Kölliker-Fuse nucleus, and in the ventral part of the lateral parabrachial nucleus. There is a striking similarity between the distribution of HRP-labeled cells in the dorsolateral pontine tegmentum of the cat and monkey and that of catecholamine-containing cells observed in this area in previous studies.     

Nuclei and subnuclei gene expression profiling in mammalian brain

Information on the neuroanatomical expression of a given gene is critical to understanding its function in the central nervous system. The integration of laser capture microdissection (LCM), T7-based RNA amplification and cDNA microarrays allows for this information to be simultaneously generated for thousands of genes. To validate this integrative approach, we catalogued the gene expression profiles of seven rat brain nuclei or subnuclei. A hundred cells from the following seven brain nuclei were analyzed: locus coeruleus (LC), dorsal raphe nucleus (DR), parvocellular division (PA) and magnocellular division (MG) of the hypothalamic paraventricular nucleus (PVN) and CA1, CA3 and dentate gyrus (DG) divisions of the hippocampal formation. Of the 2145 genes investigated, 1402 genes (65%) gave a hybridization signal statistically different from the background level that was defined by non-specific hybridizations to 15 different plant genes. Validation of our microarray data on four arbitrarily selected genes was confirmed by Real-Time PCR. Previous research showing expression patterns of 'signature' genes (n=17) for specific brain nuclei are consistent with our findings. For example, as previously shown, enriched mRNA expression encoding the serotonin transporter or tyrosine hydroxylase was found in DR and LC cells, respectively. Interestingly, expression of the serotonin 5-HT(2B) receptor mRNA was also found in DR cells. We confirmed this new finding by in-situ hybridization. The hierarchical clustering analysis of gene expression shows that the two divisions of the PVN (PA and MG) are closely related to each other, as well as the three regions of the hippocampal formation (CA1, CA3 and DG), which also showed similar gene expression profiles. This study demonstrates the importance, feasibility and utility of cellular brain nuclei profiling.     

Prenatal stress alters Fos protein expression in hippocampus and locus coeruleus stress-related brain structures

Prenatal stress (PS) durably influences responses of rats from birth throughout life by inducing deficits of the hypothalamo-pituitary-adrenal (HPA) axis feedback. The neuronal mechanisms sustaining such alterations are still unknown. The purpose of the present study was to determine whether in PS and control rats, the exposure to a mild stressor differentially induces Fos protein in hippocampus and locus coeruleus, brain areas involved in the feedback control of the HPA axis. Moreover, Fos protein expression was also evaluated in the hypothalamic paraventricular nucleus (PVN) that reflect the magnitude of the hormonal response to stress. Basal plasma corticosterone levels were not different between the groups, while, PS rats exhibited higher number of Fos-immunoreactive neurons than controls, in the hippocampus and locus coeruleus in basal condition. A higher basal expression of a marker of GABAergic synapses, the vGAT, was also observed in the hypothalamus of PS rats. Fifteen minutes after the end of the exposure to the open arm of the elevated plus-maze (mild stress) a similar increased plasma corticosterone levels was observed in both groups in parallel with an increased number of Fos-immunoreactive neurons in the PVN. Return to basal plasma corticosterone values was delayed only in the PS rats. On the contrary, after stress, no changes in Fos-immunoreactivity were observed in the hippocampus and locus coeruleus of PS rats compared to basal condition. After stress, only PS rats presented an elevation of the number of activated catecholaminergic neurons in the locus coeruleus. In conclusion, these results suggest for the first time that PS alters the neuronal activation of hippocampus and locus coeruleus implicated in the feedback mechanism of the HPA axis. These data give anatomical substrates to sustain the HPA axis hyperactivity classically described in PS rats after stress exposure.     

The development and postnatal organization of primary afferent projections to the rat thoracic spinal cord

Primary afferent projections to the thoracic spinal cord in fetal and postnatal rats were labelled by applying horseradish peroxidase (HRP) to the central stumps of cut peripheral nerves. Diaminobenzidine (DAB) and tetramethyl benzidine (TMB) histochemical processing procedures were used to reveal the HRP reaction product. In postnatal rats, individual muscle nerves were labelled to reveal the organization of muscle afferent projections to the motor nuclei. The terminals of muscle afferents were distributed widely across the dendritic arbors of motoneurons supplying the same muscles. No spatial segregation of the terminations of different populations of muscle afferents was discernable. Afferents supplying different regions of the skin were labelled by applying HRP to the dorsal and ventral primary rami of the spinal nerves. Afferents in the dorsal rami projected to lateral portions of both the ipsilateral and contralateral dorsal horns while afferents in the ventral rami projected to the medial portions of both dorsal horns. The projections of the dorsal rami were shifted caudally relative to those of the ventral rami. This relationship reflects the fact that the regions of skin innervated by the dorsal rami are displaced caudally relative to those innervated by the corresponding ventral rami. In fetuses, dorsal rami were labelled alone or in combination with ventral rami. These experiments disclosed the time course of development of the projections to different laminae of the spinal gray matter and revealed that afferents in the two primary rami project to appropriate regions in the ipsilateral and contralateral dorsal horns from the very outset.     

Afferent projections to the hypothalamic area controlling emotional responses (HACER)

The Mesulam technique for horseradish peroxidase was used to study the subcortical afferent projections to a location in the hypothalamus that has been shown to control the complete cardiovascular (CV) response accompanying a specific emotional behavior.Major projections common to all baboons injected included the lateral septal nucleus; medial, cortical and basal amygdala; the anteroventral third ventricle area; the preoptic areas; the subiculum; the paraventricular nucleus of the thalamus; periventricular gray and the central gray of the midbrain; the midbrain tegmentum; locus ceruleus, parabrachial and raphe cells in the pons; and in the medulla, raphe nuclei, the nucleus of the solitary tract, in and around the dorsal motor nucleus of the vagus, and in the region of the nucleus ambiguus. Other projections in some but not all baboons included the subfornical organ and the midline and dorsomedial nuclei of the thalamus. The nucleus of the diagonal band of Broca was labeled to some degree with all injections but was most heavily labeled with the injection extending more laterally in the hypothalamus.These results fit well with physiological and behavioral studies dealing with neural control of emotional and CV responses and support the concept of an integrative area in the hypothalamus concerned specifically with the control of CV response accompanying emotion.     

Endotoxin administration induced differential neurochemical activation of the rat brain stem nuclei

Lipopolisaccharide (LPS) is a potent activator of the immune system, but also activates the hypothalamic-pituitary-adrenocortical (HPA) axis and cerebral catecholamine systems. In the present study, the effect of peripheral LPS administration on catecholaminergic and serotonergic neurotransmission in discrete brainstem nuclei was examined. Two hours following systemic administration of LPS (1, 10, or 100 μg/kg) norepinephrine (NE) content in the locus coeruleous (LC) was significantly increased in a dose related manner. An increased dopamine (DA) turnover as reflected by the 3,4-dihydroxyphenylacetic (DOPAC) + Homovanillic acid (HVA)/DA ratio, [DOPAC + HVA]/[DA], was also observed at the LC with the medium and high doses of LPS administered. Endotoxin caused the main effects in the nucleus of the tractus solitarii (NTS) in which (a) it was found NE content increased in a dose related fashion, (b) DA turnover index was elevated with 10 and 100 μg/kg LPS doses, and (c) levels of serotonin (5-HT) and its catabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), were also significantly elevated following the injection of 10 or 100μg/kg LPS. By contrast, a consistent lack of catecholaminergic and serotonergic responses to endotoxin treatment was observed at the level of midbrain Raphe nuclei (MRN). These results demonstrate that differential neurochemical changes may occur in the brainstem region with a rank order of activation by LPS that was NTS>LC>MRN, suggesting different neural substrate for central effects of peripheral immune activation.     

Differential projections from locus coeruleus to olfactory bulb and olfactory tubercle: an HRP study

The microiontophoretic administration of horseradish peroxidase (HRP) to the olfactory bulb (OB) or olfactory tubercle (OT) in cats and rats yielded similar results in both species. After an OB HRP-injection ipsilateral and contralateral labelled neurons were seen in the piriform cortex, polymorphic layer of OT, magnocellular preoptic region, lateral hypothalamus, ventromedial hypothalamic nucleus and locus coeruleus (LC). In both species more labelled structures were found after an OT HRP-injection than after an OB HRP-injection. The substantia nigra in rats was more abundantly labelled after an OT injection than after an OB one. In cats the dorsal and the ventral raphe were also labelled. In either species, OT HRP-injections resulted in a higher frequency of LC labelled neurons than after OB injections. These results favor the hypothesis that the OT plays an important role as a relay station for efferent inflow from the brain stem en route to the OB.     

Age-dependent changes in projections from locus coeruleus to hippocampus dentate gyrus and frontal cortex

Age-dependent changes in noradrenergic innervations of the hippocampal dentate gyrus (DG) and the frontal cortex (FC) have been studied in male F344 rats. The projections from the nucleus locus coeruleus (LC) to DG or FC with advancing age (from 7 to 27 months) in rats have been quantified by electrophysiological and immunohistochemical methods. In the electrophysiological study, we observed that the percentage of LC neurons activated antidromically by electrical stimulation (P-index) of DG or FC decreased with age. We found that the percentage of LC neurons showing multiple antidromic latencies (M-index), which suggests axonal branching of individual LC neurons, increased markedly between 15 and 17 months in DG or FC. In DG, the M-index increased steadily between 15 and 24 months. In contrast, the increased M-index in FC was maintained until 24 months. The increased M-index in both targets declined at 27 months. These results suggest that LC neurons give rise to axonal branching following the loss of projections to DG or FC with age. In the immunohistochemical study, the density of dopamine-beta-hydroxylase-positive axonal varicosities was measured in molecular, granule cell and polymorphic layers of DG. The density in the polymorphic layer significantly decreased in the earlier stage of ageing (7-19 months), whilst the density in the molecular and granule cell layers decreased in the later stage (27 months). These findings suggested that a layer-specific decline occurred with age in the noradrenergic axon terminals in DG.     

Nitric oxide synthase in the hypothalamic paraventricular nucleus of the female rat; organization of spinal projections and coexistence with oxytocin or vasopressin

We investigated the distributions and interrelations of neuronal nitric oxide (NO) synthase- (nNOS), oxytocin- (OT), and 8-arginine vasopressin- (AVP) immunoreactive (IR) neurons in the paraventricular nucleus (PVN), and the occurrence and distribution of nNOS spinally projecting neurons in the PVN of the female rat. Using double labelling immunohistochemistry, we mapped the distribution of nNOS-, OT- and AVP-immunoreactive (IR) neuronal cell bodies in the different parts of the PVN. About 80% of nNOS-IR cell bodies were magnocellular. About 30% of the nNOS-IR cell bodies were OT-IR, colocalization being most frequent in the rostral parts. In comparison, only approximately 3% of all nNOS-IR cell bodies were AVP-IR, evenly distributed throughout the PVN. True Blue (TB), administered unilaterally into the spinal cord, disclosed that most spinally projecting cell bodies in the PVN were localized in caudal parts. Combined TB tracing and nNOS immunohistochemistry showed that approximately 30% of spinally projecting neurons in the PVN were nNOS-IR, and that approximately 40% of these were magnocellular. Ipsilateral nNOS spinal projections were about eight times more frequent than the contralateral nNOS projections. The study describes the detailed neuroanatomical organization of nNOS neurons coexpressing OT or AVP, and of nNOS spinally projecting neurons within defined parts of the PVN. In contrast to the paraventriculo-spinal system in general, we show that the nNOS paraventriculo-spinal pathway to a large extent originates in magnocellular cell bodies. The results suggest that NO is an important messenger in the paraventriculo-spinal pathway that may in part act in concert with OT.     

Postmortem tracing reveals the organization of hypothalamic projections of the suprachiasmatic nucleus in the human brain

The suprachiasmatic nucleus (SCN) is a small structure considered to be the site of the major circadian pacemaker of the mammalian brain. Disturbances in human biological clock function may occur in several diseases, such as Alzheimer's disease, sleep problems, and seasonal depression. Since basic knowledge of the anatomical connections of the human SCN is limited due to the lack of suitable neuroanatomical tracing methods, the understanding of physiological mechanisms of human SCN function has obviously been hampered. In the present study, the hypothalamic connections of the human SCN were revealed for the first time with a newly developed in vitro postmortem anterograde tracing method. The human SCN was found to be connected with nuclei in the hypothalamus that are involved in hormone secretion, cardiovascular regulation, and behavior activity. These human SCN projections appear to follow the same general patterns as those in the rodent brain. This homology may indicate an evolutionary conservation of the SCN projections from rodent to human. Through these connections, the human SCN may transmit its circadian information to regulate hormone secretion, body temperature, and behavioral functions as it does in animal species. In addition, the postmortem tracing technique may be a valuable tool that will contribute to our understanding of anatomical connections in the human brain, and may have other applications in the research on the physiology and pathology of the human brain. J. Comp. Neurol. 400:87–102, 1998. © 1998 Wiley-Liss, Inc.