Hypothalamic galanin-immunoreactive neurons projecting to the posterior lobe of the rat pituitary: a combined retrograde tracing and immunohistochemical study.

To identify the galanin-immunoreactive neurons projecting to the posterior lobe of the pituitary in the rat hypothalamus, a retrograde tracer (complex of wheat germ agglutinin-enzymatically inactive horseradish peroxidase-colloidal gold) was injected into the posterior lobe of the pituitary. Sections of the hypothalamus were treated with a combination of silver enhancement of retrogradely transported tracer and immunohistochemistry of galanin. Of the total number of hypothalamic cells doubly labeled with retrograde tracing and galanin-immunostaining, 56-60% were found in the supraoptic nucleus, 18-23% in the retrochiasmatic nucleus, 8-10% in the lateral magnocellular portion of the paraventricular nucleus. The ratio of (number of doubly labeled cells/number of galanin-immunoreactive cells) in each of the above regions was similar to the ratio of (number of retrogradely labeled cells/number of Nissl-stained cells) in the supraoptic nucleus. Of all retrogradely labeled cells in the hypothalamus, 51-56% also contained galaninlike immunoreactivity. In conclusion: (1) galanin-immunoreactive fibers in the posterior lobe of the pituitary originate mainly in the supraoptic nucleus, retrochiasmatic nucleus, and lateral magnocellular portion of the paraventricular nucleus, (2) most of galanin-immunoreactive cells in these regions project to the posterior lobe of the pituitary, and (3) about half the neurons constituting the hypothalamo-neurohypophyseal system contain galaninlike immunoreactivity.     

Localization of forebrain neurons which project directly to the medulla and spinal cord of the rat by retrograde tracing with wheat germ agglutinin

Wheat germ agglutinin (WGA) in a slow-release polyacrylamide gel pellet was implanted in the medulla or spinal cord of the rat. Large numbers of retrogradely labeled cells were visualized by immunocytochemical procedures in specific nuclei of the forebrain mainly ipsilateral to the implant site following implants as far caudal as the sacral segments of the spinal cord. Total average number of labeled forebrain cells (three brains per category; 100 μm per 150 μm of brain tissue were examined microscopically): medulla, 2,115; cervical, 1,878; lumbar, 1,017; sacral, 385. After WGA-gel implants in the medulla or cervical cord the majority of retrogradely labeled neurons were seen in the lateral hypothalamic area, the zona incerta, and in subdivisions, of the paraventricular nucleus. A continuum of labeled cells extended from the caudal part of the paraventricular nucleus into the posterior hypothalamus and into the central gray of the midbrain. Labeled cells were also seen in the medial basal hypothalamus and the rostral part of the bed nucleus of the stria terminalis. A few labeled cells were observed in the medial and lateral preoptic areas, the rostral part of the paraventricular nucleus, and in the arcuate nucleus. Following WGA-gel implants in the lumbar or sacral cord many retrogradely labeled cells were observed mainly in the paraventricular nucleus, the lateral hypothalamus, zona incerta, medial basal hypothalamus, and posterior hypothalamic area. The continuum of labeled cells described above was also seen following these implants. Our data indicate that the lateral hypothalamus and zona incerta, as well as specific parts of the paraventricular nucleus, are major loci of neurons which project directly to the medulla and spinal cord of the rat. The consistency with which labeled cells were localized across all brains examined within categories of implant sites and the large numbers of labeled cells counted within these areas appeared to verify the sensitivity of our retrograde tracing method. Therefore, we interpret the paucity or absence of labeled cells in particular brain regions to indicate that cells of these regions do not project to the medulla or spinal cord.     

Direct Link From the Suprachiasmatic Nucleus to Hypothalamic Neurons Projecting to the Spinal Cord: A Combined Tracing Study Using Cholera Toxin Subunit B and Phaseolus vulgaris-Leucoagglutinin

By combining retrograde and anterograde tracing, evidence for a bineuronal connection from the suprachiasmatic nucleus (SCN) to the intermediolateral cell column in the spinal cord (IML) was obtained. The retrograde tracer cholera toxin subunit B (ChB) was pressure-injected into the spinal cord and the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) was iontophoretically injected into the SCN. The two tracers were visualized simultaneously by a double immunohistochemical procedure. In the hypothalamus, ChB injections gave rise to retrogradely labeled cell bodies in the paraventricular nucleus, retrochiasmatic area, perifornical region, lateral hypothalamic area, and the posterior hypothalamic area. The SCN were found to project to all of these areas. Furthermore, spinal-projecting neurons were found in the brain stem, but no efferents from the SCN were observed to innervate these areas. In the most sparsely innervated areas, the lateral hypothalamic area and the perifornical region, only occasionally a PHA-L fiber in close apposition to a ChB-ir cell body was observed. This was also the case in the retrochiasmatic area and posterior hypothalamic area, although these areas received a moderate number-immunoreactive (ir) PHA-L-ir fibers. The highest number of closely apposed PHA-L-ir fibers and ChB-ir cell bodies was observed in the dorsal parvicellular and in the ventral division of the medial parvicellular paraventricular nucleus, which were also the areas receiving the densest input from the SCN. By anterograde tracing from the paraventricular nucleus of the hypothalamus, the exact topography of the terminal field formed by descending paraventricular neurons was established. Thus, it was confirmed that the paraventricular nucleus of the hypothalamus predominantly innervates the IML. The present study suggests the existence of a bineuronal link between the SCN and the IML, possibly involved in transmission of circadian signals from the endogenous clock to the pineal gland and other organs receiving sympathetic afferents.     

Bed nucleus of the stria terminalis: cytoarchitecture, immunohistochemistry, and projection to the parabrachial nucleus in the rat

The bed nucleus of the stria terminalis (BST) sends a dense projection to the parabrachial nucleus (PB) in the pons. The BST contains many different types of neuropeptidelike immunoreactive cells and fibers, each of which exhibits its own characteristic distribution within cytoarchitecturally distinct BST subnuclei. Corticotropin releasing factor (CRF)-, neurotensin (NT)-, somatostatin (SS)-, and enkephalin (ENK)-like immunoreactive (ir) neurons are particularly numerous within areas of the BST that project to the PB. In this study, we use the combined retrograde fluorescence-immunofluorescence method to determine whether neurons in the BST that project to the PB contain these immunoreactivities. After Fast Blue injections into PB, retrogradely labeled neurons were numerous throughout the lateral part of the BST, particularly in the dorsal lateral (DL) and posterior lateral subnuclei. Retrogradely labeled neurons were also present in the preoptic, ventral lateral, and supracapsular BST subnuclei and in the parastrial nucleus. Many of the CRF-ir, NT-ir, and SS-ir neurons in DL were retrogradely labeled. A few double-labeled cells of each type were also found in the posterior lateral, ventral lateral and supracapsular BST subnuclei ENK-ir neurons were never retrogradely labeled. Our results show that BST neurons that project to the PB stain for the same neuropeptides as those in the central nucleus of the amygdala (CeA) that project to the PB, demonstrating further the close anatomical relations between these two structures.     

Organization of cortical, basal forebrain, and hypothalamic afferents to the parabrachial nucleus in the rat.

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

Collateral axonal projections from hypothalamic hypocretin neurons to cardiovascular sites in nucleus ambiguus and nucleus tractus solitarius

Hypocretin-1 (hcrt-1)-containing axons have been shown to have an extensive distribution within the central nervous system, although the total number of hypothalamic hcrt-1 neurons has been shown to be small. This suggests that hcrt-1 neurons may innervate central structures with similar function through collateral axonal projections. Retrograde tract-tracing techniques combined with immunohistochemistry were used in this study to investigate whether hypothalamic hcrt-1-containing neurons send collateral axonal projections to cardiovascular sites in the nucleus of the solitary tract (NTS) and in the nucleus ambiguus (Amb) in the rat. Fluorogold- (FG) and/or rhodamine (Rd)-labeled latex microspheres were microinjected into either the NTS or Amb at sites that elicited bardycardia responses (L-glutamate; 0.25 M; 10 nl). After a survival period of 10-15 days, the rats were sacrificed and tissue sections of the hypothalamus were processed immunohistochemically for the identification of hcrt-1-containing cell bodies. After injection of the tract-tracers into the NTS or Amb, retrogradely labeled neurons were observed within several hypothalamic regions; the paraventricular hypothalamic nucleus, lateral hypothalamic area, perifornical hypothalamic area, and posterior hypothalamus, bilaterally, but with an ipsilateral predominance. In addition, after NTS injections, retrogradely labeled neurons were found within the ipsilateral caudal arcuate nucleus. Of the total number (1107+/-97) of hcrt-1-immunoreactive neurons found bilaterally within the lateral and perifornical hypothalamic nuclei, 7.9+/-1.4% were found to be retrogradely labeled from the NTS, 16.4+/-1.8% from the Amb, and 3.1+/-0.5% from both medullary sites. Hcrt-1 neurons projecting to the NTS were found mainly in and around the perifornical hypothalamic region, with a smaller number in the caudal lateral hypothalamic area. On the other hand, those innervating the Amb were primarily observed within the caudal lateral hypothalamic area, with a smaller number in the perifornical hypothalamic area. Neurons with collateral axonal projections to NTS and Amb were observed within two specific hypothalamic areas: one group of neurons was found in the perifornical hypothalamic area, and the other was observed in the lateral hypothalamic region just dorsal to the retrochiasmatic component of the supraoptic nucleus. These data indicate that axons from hcrt-1 neurons bifurcate to innervate functionally similar cardiovascular-responsive sites in the NTS and Amb. Although the function of these hcrt-1-containing hypothalamic-medullary pathways is not known, they likely represent the anatomical substrate by which the lateral hypothalamic hcrt-1 neurons simultaneously coordinate autonomic-cardiovascular responses to different behaviors.     

Evidence for corticotropin-releasing hormone projections from Barrington's nucleus to the periaqueductal gray and dorsal motor nucleus of the vagus in the rat

The present study used anterograde and retrograde tract tracing and immunohistochemistry to determine the efferent projections of corticotropin-releasing hormone (CRH) neurons of Barrington's nucleus in the rat. Injections of Phaseolus vulgaris-leucoagglutinin into Barrington's nucleus resulted in anterograde labeling in the dorsal motor nucleus of the vagus, periaqueductal gray, medial thalamic nuclei, lateral hypothalamus, paraventricular nucleus of the hypothalamus, lateral preoptic area, and lateral septum. The retrograde tract tracer, fluorogold, injected into the lumbosacral spinal cord labeled many, but not all, CRH-immunoreactive neurons in Barrington's nucleus. Moreover, some Barrington's neurons that were retrogradely labeled from the spinal cord were not CRH-immunoreactive. Several CRH-immunoreactive Barrington's neurons were retrogradely labeled by fluorogold injections into the periaqueductal gray, and these were located predominantly in the dorsal part of the nucleus. Additionally, some CRH-immunoreactive Barrington's neurons were retrogradely labeled from fluorogold injections into the dorsal motor nucleus of the vagus. In contrast, fluorogold injections into the lateral hypothalamus, lateral preoptic area, or lateral septum did not result in double labeling of CRH-immunoreactive neurons in Barrington's nucleus. These results suggest that many, but not all, CRH-containing neurons of Barrington's nucleus project to the lumbosacral spinal cord. In addition to their previously documented projections to the spinal cord, these neurons may be a source of CRH in the periaqueductal gray and dorsal motor nucleus of the vagus. CRH projections of Barrington's nucleus may play a role in behavioral or autonomic aspects of stress responses, in addition to their proposed role in micturition. © 1995 Wiley-Liss, Inc.     

Neurotensin in the rat median eminence: the possible sources of neurotensin-like fibers and varicosities in the external layer

The possible sources of neurotensin-like immunoreactive axons in the median eminence were studied after several experimental surgical approaches including unilateral lateral retrochiasmatic area transection, midsagittal knife cut through the median eminence, complete surgical isolation of the medial basal hypothalamus and bilateral paraventricular nucleus lesions. Both immunohistochemical and radioimmunoassay data demonstrate that neurotensin-containing neuronal located in the hypothalamic arcuate nuclei represent the main source of neurotensin occurring in the external zone of the median eminence of the rat: (1) neither the complete isolation of the medial basal hypothalamus nor the transection of the major neuronal input channel to the median eminence in the lateral retrochiasmatic area altered neurotensin-like immunoreactivity in the median eminence; (2) bilateral lesioning of the paraventricular nucleus resulted in insignificant changes of neurotensin level in the median eminence; and (3) two days after lesioning the median eminence an increased amount of retrogradely accumulated neurotensin-like immunoreactivity was found in several perikarya of the arcuate nuclei due to the blockage of axonal transport in the transected fibers. Retrograde accumulation of neurotensin-like material in other cells scattered in the anterior hypothalamus (in the paraventricular, paraventricular and anterior hypothalamic nuclei) indicates that in addition to the arcuate neurons these neurons may also participate in the neurotensin innervation of the median eminence.     

Efferent connections from the lateral hypothalamic region and the lateral preoptic area to the hypothalamic paraventricular nucleus of the rat

The lateral preoptic and lateral hypothalamic regions contain the majority of the cell groups embedded in the fibre trajectories of the medial forebrain bundle on its course through the hypothalamus. Recent studies have extended considerably the parcellation of the lateral hypothalamic region, and, therefore, the need to emphasize new insights into the anatomical organisation of projections from the neurons of the lateral hypothalamic region. In the present study we describe the anatomical organisation of efferent projections from the lateral preoptic and lateral hypothalamic regions to the hypothalamic paraventricular nucleus (PVN) on the basis of retrograde- and anterograde-tracing techniques. Iontophoretic injections of the retrograde tracer, cholera toxin subunit B, into the PVN revealed that most hypothalamic nuclei project to the PVN. Within the lateral hypothalamic region, retrogradely labelled cells were concentrated in the intermediate hypothalamic area, the lateral hypothalamic area, and the perifornical nucleus, whereas fewer retrogradely labelled cells were found in the lateral preoptic area. To determine the distribution of terminating fibres in subnuclei of the heterogeneous PVN, iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin were delivered into distinct areas of the lateral hypothalamic region. Neurons of the intermediate hypothalamic area projected mainly to the PVN subnuclei, which contained parvicellular neuroendocrine cells. In contrast, neurons of the rostral and tuberal parts of the lateral hypothalamic area and the perifornical nucleus projected to the PVN subnuclei, which contained parvicellular neurons that send descending projections to preganglionic cell groups in the medulla and spinal cord. The perifornical nucleus was the only area within the lateral hypothalamic region that consistently innervated magnocellular perikarya of the PVN. Finally, all areas of the lateral hypothalamic region contributed substantially to fibres terminating in the perinuclear shell of the PVN. These results demonstrate that anatomically distinct areas of the lateral hypothalamic region have distinct projections to subnuclei of the PVN and further substantiate the view that the lateral hypothalamic region as well as the PVN constitute anatomically and functionally heterogeneous structures. © 1994 Wiley-Liss, Inc.     

CNS connections with the median raphe nucleus: retrograde tracing with WGA-apoHRP-Gold complex in the rat

In this work we examined the neuronal input to one of the serotoninergic centers in the brain, median raphe nucleus (MR). Special consideration is given to projections of the hypothalamus. To describe the afferents to MR, a retrograde transport technique was used after microinjection of WGA-apoHRP-Gold complex under pressure and subsequent gold-silver intensification on formaldehyde-fixed rat brain sections. Optimal conditions were obtained when the coordinates of the injection site were A +/- 1.5, L +/- 0.15, and H +/- 2.7 according to Paxinos and Watson (The Rat Brain in Stereotaxic Coordinates. New York: Academic Press, '82). Results obtained under these conditions show a heterogeneous distribution of labeled neurons throughout the brain, including a large proportion (+/- 65%) of hypothalamic neurons. Extra-hypothalamic neurons projecting to MR were from the prefrontal cortex, lateral and medial habenular nuclei, the pontine area of the central grey, interpeduncular nucleus, dorsal raphe nucleus, oculomotor and trochlear nuclei, dorsal and laterodorsal tegmental nuclei, parabrachial nuclei, and lateral and interpositus cerebellar nuclei. Hypothalamic neurons connected to MR were found to be from medial and lateral preoptic areas, lateral hypothalamus, dorsomedian nucleus, the perifornical area, and the complex of mammillary bodies. Many other discrete regions contained different densities of labeled perikarya: the medial preoptic nucleus, paraventricular nucleus, retrochiasmatic area, arcuate nucleus, lateral magnocellular nucleus, and the posterior area. The MR appears as an integrative center receiving many neuroanatomically and functionally heterogeneous inputs from the whole brain.     

Neuronal projections to the medial preoptic area of the sheep,with special reference to monoaminergic afferents: Immunohistochemical and retrograde tract tracing studies

The preoptic area contains most of the luteinizing hormone releasing hormone immunoreactive neurons and numerous monoaminergic afferents whose cell origins are unknown in sheep. Using tract tracing methods with a specific retrograde fluorescent tracer, fluorogold, we examined the cells of origin of afferents to the medial preoptic area in sheep. Among the retrogradely labeled neurons, immunohistochemistry for tyrosine hydroxylase, dopamine-β-hydroxylase, phenylethanolamine N-methyltransferase, and serotonin was used to characterize catecholamine and serotonin fluorogold labeled neurons. Most of the afferents came from the ipsilateral side to the injection site. It was observed that the medial preoptic area received major inputs from the diagonal band of Broca, the lateral septum, the thalamic paraventricular nucleus, the lateral hypothalamus, the area dorsolateral to the third ventricle, the perimamillary area, the amygdala, and the ventral part of the hippocampus. Other numerous, scattered, retrogradely labeled neurons were observed in the ventral part of the preoptic area, the vascular organ of the lamina terminalis, the ventromedial part of the hypothalamus, the periventricular area, the area lateral to the interpeduncular nucleus, and the dorsal vagal complex. Noradrenergic afferents came from the complex of the locus coeruleus (A6/A7 groups) and from the ventro-lateral medulla (group A1). However, dopaminergic and adrenergic neuronal groups retrogradely labeled with fluorogold were not observed. Serotoninergic fluorogold labeled neurons belonged to the medial raphe nucleus (B8, B5) and to the serotoninergic group situated lateral to the interpeduncular nucleus (S4). In the light of these anatomical data we hypothesize that these afferents have a role in the regulation of several functions of the preoptic area, particularly those related to reproduction. Accordingly these afferents could be involved in the control of luteinizing hormone releasing hormone (LHRH) pulsatility or of preovulatory LHRH surge.     

Enkephalin-immunoreactive neurons in the parvicellular subdivisions of the paraventricular nucleus project to the external zone of the median eminence.

The enkephalin-immunoreactive neurons that project to the external zone of the median eminence were identified on thin paraffin and thick vibratome sections using a combination of retrograde labeling with peripherally administered Fluoro-Gold and immunocytochemistry. The vast majority of the enkephalin-immunoreactive neurons that project to the external zone of the median eminence (ME) reside in the paraventricular nucleus (PVN) of the hypothalamus. Within the PVN, the majority of these hypophysiotropic neurons are located in the medial parvicellular subdivision, while a smaller number can be detected in the anterior and the periventricular subdivisions. Although many enkephalin-immunoreactive neurons are present in other hypophysiotropic areas of the hypothalamus, such as the medial preoptic area, the anterior periventricular area, and the arcuate nucleus, only a few of these can be retrogradely labeled from the ME. These results provide morphological evidence for the key role of paraventricular enkephalin-immunoreactive neurons in the regulation of neuroendocrine functions.     

CNS projections to the pterygopalatine parasympathetic preganglionic neurons in the rat: a retrograde transneuronal viral cell body labeling study.

The retrograde transneuronal viral cell body labeling method was used to study the CNS nuclei that innervate the parasympathetic preganglionic neurons which project to the pterygopalatine ganglion. Small injections of a suspension of pseudorabies virus (PRV) were made in the pterygopalatine ganglion of rats and after 4 days their brains wer e processed for immunohistochemical detection of PRV. Some of the tissues were stained with a dual immunofluoresence method that permitted the visualization of PRV and neurotransmitter enzyme or serotonin immunoreactivity in the same cell. Retrograde cell body labeling was detected in the ipsilateral ventrolateral medulla oblongata in the region that has been termed the superior salivatory nucleus. This area was the same region that was retrogradely labeled after Fluoro-Gold dye injections in the pterygopalatine ganglion. Retrograde transneuronally infected cell bodies that provide putative afferent inputs to the pytergopalatine parasympathetic preganglionic neurons were mapped throughout the brain. In the medulla oblongata, transneuronally labeled neurons were seen in the nucleus tractus solitarii, dorsomedial part of the spinal trigeminal nucleus and gigantocellular reticular nucleus. In most experiments, some A1 catecholamine cells and serotonin neurons of the raphe magnus, raphe pallidus, raphe obscurus, and parapyramidal nuclei were labeled. In the pons, labeled cells were found in the parabrachial nucleus. A5 catecholamine cell group, and non-catecholamine part of the subcoeruleus region. In the midbrain, cell body labeling was located in the central gray matter and retrorubral field. In the diencephalon, labeling was found mainly in the hypothalamus. The areas included the lateral hypothalamic area, lateral preoptic area, dorsomedial and paraventricular hypothalamic nuclei, and ventral zona incerta. Contralateral second order cell body labeling was seen in the tuberomammillary nucleus of the hypothalamus. Some of these cells were histidine decarboxylase-immunoreactive. In the forebrain, the bed nucleus of the stria terminalis, substantia innominata, and an area of the cerebral cortex called the amygdalopiriform transition zone were labeled.     

Afferent connections of the hypothalamic retrochiasmatic area in the rat

The afferent projections to the retrochiasmatic area (RCA) of the rat hypothalamus have been studied by means of retrograde axonal transport of horseradish peroxidase. Iontophoretic deposit of the marker was used in most animals, and consistent projections from the hypothalamic ventromedial nucleus, the lateral part of the substantia nigra and the parabigeminal nucleus (NPB) were observed. A comparison between NPB projections and projections from the neighboring tegmentum suggests that some neurons of the NPB project to both the RCA and the superior colliculus. Consequently, these NPB neurons might link visual information on its way toward the retrochiasmatic area which, in view of its strategic position, could play a role in neuroendocrine processes.     

Immunohistochemical localization of avian pancreatic polypeptide-like immunoreactivity in the rat hypothalamus

The distribution of avian pancreatic polypeptide-like (APP) immunoreactivity within the rat hypothalamus was investigated with the indirect immunoperoxidase method. APP immunoreactive perikarya are found in largest numbers in the retrochiasmatic area, the arcuate nucleus, and the supracommissural portion of the interstitial nucleus of the stria terminalis. Small clusters of immunoreactive neurons are also consistently observed in the ventral aspect of the medial preoptic area and lateral hypothalamic area, immediately dorsolateral to the optic chiasm and tracts. These neurons are apparent in all animals but are more intensely strained and occur in larger numbers following colchicine pretreatment. Other immunoreactive neurons are visible only in colchine-treated rats and are scattered throughout the anterior and lateral hypothalamic areas and the supramammillary nucleus. Immunoreactive axons and terminal fields present an extensive and highly characteristic distribution throughout the hypothalamus, which in many instances exhibits differential distribution within specific subfields of hypothalamic nuclei and areas. The heaviest concentrations of APP immunoreactive axons are present in the periventricular nucleus throughout the rostrocaudal extent of the hypothalamus, the ventrolateral portion of the suprachiasmatic nucleus, the retrochiasmatic area, the parvocellular paraventricular nucleus, the ventral supraoptic nucleus, the perifornical nucleus, the ventral dorsomedial nucleus, and the arcuate nucleus. Moderate plexuses of immunoreactive fibers are also present in the medial preoptic area, the anterior and lateral hypothalamic areas, the nucleus circularis, the median eminence, and the ventral premammillary area. Other areas, such as the ventromedial nucleus, contain virtually no immunoreactive axons but are encapsulated by a dense plexus of immunoreactive terminals. The distribution of a major component of APP immunoreactive fibers exhibits a marked similarity to that of previously described norepinephrine-containing hypothalamic afferents. Other groups of APP immunoreactive perikarya and fibers appear to represent components of intrinsic diencephalic systems.     

Differences in hypothalamic Fos expressions between two heat stress conditions in conscious mice

Hyperthermia and dehydration were important physiological phenomena in heat stress. But, the degrees of these phenomena were changed by heat stress conditions, and the distinction between both phenomena is necessary for investigation of response for individual phenomenon. Heat stress at 34 degrees C for 60 min increased rectal temperature, and heat stress at 38.5 degrees C for 60 min further increased rectal temperature and increased osmolality in mice. We investigated the activated region in hypothalamus, which played a role in thermoregulation, fluid regulation and so on, using immunostaining for Fos protein under these conditions in conscious mice. At 34 degrees C, Fos-positive neurons increased in the median preoptic nucleus, lateral preoptic area and anterior hypothalamic area, which were known to be the thermoregulatory center, and the dorsomedial hypothalamic nucleus, which was known to control eating. At 38.5 degrees C, Fos-positive neurons further increased in the regions mentioned above and appeared in the lateral septal nucleus, medial preoptic area, lateral hypothalamic area and zona incerta, which were thought to be involved in thermoregulation and/or fluid regulation, and the paraventricular hypothalamic nucleus, supraoptic nucleus and supraoptic nucleus in the retrochiasmatic part, which were known to be involved in neuroendocrine effector systems. These results support that the activated regions in hypothalamus differed with heat stress conditions, which induced only hyperthermia and both hyperthermia and dehydration.     

Most neurons in the nucleus tractus solitarii do not send collateral projections to multiple autonomic targets in the rat brain

The nucleus tractus solitarii (NTS) receives primary visceral afferents and sends projections to other autonomic nuclei at all levels of the neuroaxis. However, it is unknown if distinct populations of NTS neurons project to individual autonomic targets or if individual neurons in the NTS project to multiple autonomic targets. Understanding the basic circuitry of visceral reflex pathways is essential for the analyses of functional central autonomic networks. We examined projections from the NTS to autonomic targets within the hypothalamus (paraventricular nucleus, PVN), pons (parabrachial nucleus, PB), and medulla (caudal ventrolateral medulla, CVL) using retrograde tracing and immunohistochemistry. Dual retrograde tracer microinjections were made into pairs of targets (PVN + CVL; PVN + PB; PB + CVL), and the pattern of retrograde labeling was examined within NTS. The extent of collateralization, seen as dual retrogradely labeled neurons, was negligible for combined PVN and CVL injections and increased for injections combining PB with either PVN or CVL, but the majority of NTS neurons project to only one autonomic target. Immunohistochemistry for tyrosine hydroxylase (TH) was used to examine the pattern of TH-immunoreactivity (TH-ir) within retrogradely labeled NTS neurons. TH-ir was seen predominantly in projections to PVN, to a lesser degree in projections to PB, and was largely absent from projections to CVL. The percentage of dual retrogradely labeled neurons displaying TH-ir corresponded to the target displaying the most TH-ir, and TH-ir was not predictive of collateralization. Together, these results indicate that NTS neurons project to individual autonomic targets in the brain.     

An HRP study of the connections of the subfornical organ of the rat

The connections of the subfornical organ (SFO) wer investigated by using the HRP technique. Injections into the SFO labeled neurons in the medial septum, but not in lateral septum nor in the diagonal band nucleus. Labeled cells were observed in the median preoptic nucleus, below the ependyma of the third ventricle, in the dorsal preoptic region near the anterior commissure, and diffusely throughout the medial preoptic and anterior bypothalamic areas. Fibers were followed from the ventral stalk of the SFO. Precommissural fibers enter the median preoptic nucleus where many of them appear to terminate. Others continue on to the medial septum, the OVLT, the supraoptic nucleus, and the suprachiasmatic nucleus, HRP injections into the median preoptic nucleus labled many neurons in the SFO. Postcommissural fibers reach the hypothalamus by descending along the walls of the ventricle in the subependymal space, by traveling in the columns of the fornix and the medial corticohypothalamic tract, or by passing through the paraventricular nucleus of the thalamus. Some postcommissural fibers turn rostrally and enter the median preoptic nucleus while others join precommissural fibers bound for the supraoptic nucleus. More caudally directed fibers appear to innervate the paraventricular nucleus of the hypothalamus and the medial preoptic and anterior hypothalamic areas. HRP injections into the paraventricular nucleus of the hypothalamus labeled neurons in the SFO. These finding corroborate and extend previous work in describing neural connections between two brain regions that are important for fluid blance.     

Intracerebroventricular injection of senktide-induced Fos expression in vasopressin-containing hypothalamic neurons in the rat

Intracerebroventricular injection of senktide, a selective agonist for neurokinin B receptor (NK3), induced Fos expression in many neurons of the rat hypothalamus. Fos-positive neurons were predominantly present in the supraoptic and paraventricular hypothalamic nuclei, and some of them were seen in the lateral preoptic area, lateral hypothalamic area, arcuate nucleus, perifornical region, posterior hypothalamic area, circular nucleus, and along relatively large blood vessels (lateral hypothalamic perivascular nucleus) in the anterior hypothalamus. A double labeling study was performed to examine if vasopressin-containing neurons in the hypothalamus could be activated by the treatment. Neurons with both Fos-like immunoreactivity (-LI) and vasopressin-LI were found in the paraventricular nucleus, supraoptic nucleus, circular nucleus and lateral hypothalamic perivascular nucleus. In the supraoptic nucleus, about 87% of vasopressin-containing neurons exhibited Fos-LI, which corresponded to about 64% of Fos-positive neurons in the nucleus. In the paraventricular nucleus, about 80% of vasopressin-like immunoreactive neurons exhibited Fos-LI, which constituted about 51% of the total population of Fos-positive neurons in the region. The results suggest that NK3 receptor may be involved in the modulation of release of vasopressin from the hypothalamus in the rat.