Yasuhiko Hosoya

Distribution of orexin neurons in the adult rat brain

Orexin (ORX)-A and -B are recently identified neuropeptides, which are specifically localized in neurons within and around the lateral hypothalamic area (LHA) and dorsomedial hypothalamic nucleus (DMH), the regions classically implicated in feeding behavior. Here, we report a further study of the distribution of ORX-containing neurons in the adult rat brain to provide a general overview of the ORX neuronal system. Immunohistochemical study using anti-ORX antiserum showed ORX-immunoreactive (ir) neurons specifically localized within the hypothalamus, including the perifornical nucleus, LHA, DMH, and posterior hypothalamic area. ORX-ir axons and their varicose terminals showed a widespread distribution throughout the adult rat brain. ORX-ir nerve terminals were observed throughout the hypothalamus, including the arcuate nucleus and paraventricular hypothalamic nucleus, regions implicated in the regulation of feeding behavior. We also observed strong staining of ORX-ir varicose terminals in areas outside the hypothalamus, including the cerebral cortex, medial groups of the thalamus, circumventricular organs (subfornical organ and area postrema), limbic system (hippocampus, amygdala, and indusium griseum), and brain stem (locus coeruleus and raphe nuclei). These results indicate that the ORX system provides a link between the hypothalamus and other brain regions, and that ORX-containing LHA and DMH neurons play important roles in integrating the complex physiology underlying feeding behavior.

Brainstem projections from the lateral hypothalamic area in the rat, as studied with autoradiography

Descending projections from the lateral hypothalamic area to the brainstem were studied, using [3H]-amino acid autoradiography, in the rat. Two main ipsilateral paths were reorganized. One is the periventricular fiber system projecting to the midbrain central gray. The other is a fiber system which eventually descends the central tegmental field, projecting strongly to the dorsal raphe nucleus, medial and lateral parabrachial nuclei, nucleus reticularis parvocellularis, solitary nuclei and dorsal motor nucleus of the vagus nerve. Sparse projections were observed to the nuclei raphe magnus, obscurus and pallidus, group B3 (or the ventrolateral subpial group) and spinal trigeminal nucleus.

Identification and distribution of the spinal and hypophyseal projection neurons in the paraventricular nucleus of the rat. A light and electron microscopic study with the horseradish peroxidase method.

SummaryThe distribution of labeled neurons in the paraventricular nucleus of the hypothalamus (PVN) was studied following injections of horseradish peroxidase (HRP) into the spinal cord (C8 to T1) or the hypophysis in the rat. Injections were also made in the spinal cord in another group of animals, which were subjected to water deprivation for a period of 3 days, and the PVN of these animals was examined with the electron microscope.Spinal projection neurons (paraventriculospinal tract, PVST, neurons) formed two groups; the dorsal and the ventral groups. They were located within the parvocellular part of the PVN and fused into one at the caudal level. The neurons of the dorsal group were well assembled whereas those of the ventral group were intermingled with paraventriculohypophyseal tract (PVHT) neurons, which were concentrated in the magnocellular part. Electron microscopic observations revealed that HRP-labeled neurons after spinal injections did not contain neurosecretory granules and that they were not affected by water deprivation. On the other hand, neurons containing a number of neurosecretory granules displayed a significant degree of dilatation of the endoplasmic reticulum as the result of water deprivation. These neurons contained no HRP granules.The present findings suggest that the PVST neurons are distinct from the PVHT neurons and that the neuronal groups of both systems form different cell columns within the nucleus.

Descending input from the hypothalamic paraventricular nucleus to sympathetic preganglionic neurons in the rat

SummaryThe descending projection of the hypothalamic paraventricular nucleus (PVN) to the sympathetic preganglionic neurons (SPNs) in the upper thoracic cord of the rat was studied. PVN-fibers were labeled by anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L), while SPNs were retrogradely labeled with cholera toxin subunit B (CTb) which was injected into the superior cervical ganglion. SPNs labeled with CTb were mainly observed in the nucleus intermediolateralis (IML) pars principalis and pars funicularis, and a small number of them were in the nucleus intercalatus (IC) and central autonomic nucleus (CA). SPNs found in the IML had dendrites that projected in various directions. Five types of dendritic projections were noted: medial, rostral, caudal, lateral (including dorsolateral) and ventral. Longitudinal dendritic bundles interconnected each cell cluster in the IML. Medial dendrites of the IML, together with dendrites of the IC and CA, formed transverse dendritic bundles extending from the IML to the central canal. The transverse dendritic bundles disentangled near the midline and formed a loose dendritic plexus in the region just dorsal to the central canal. PVN-fibers labeled with PHA-L were observed primarily in lamina I and intermediate gray (lamina VII). Although varicose PVN-fibers and SPNs coexisted in the IML, the tight packing of the dendritic bundles prevented any clear demonstration of direct contacts between them. On the other hand, PVN-fibers were occasionally found to appose and wind around the primary or secondary dendrites of some SPNs of the CA and IC. These dendrites were studded with varicosities of PVN-fibers for a short length, and terminal boutons of PVN-fibers were also seen to make contact directly with the dendrites. The results of this study substantiated a direct connection between the PVN and SPNs, using a combination of immunohistochemical techniques for PHA-L and CTb. The possible involvement of a direct pathway from the PVN to SPNs in cardiovascular regulation is discussed.

Cells of origin of the spinocerebellar tract in the rat, studied with the method of retrograde transport of horseradish peroxidase.

Following injections of horseradish peroxidase into the cerebellum, the distribution of labeled neurons was studied in the whole length of the spinal cord of the rat. To find the ascending side of the axons, injections were made following hemisections at C1 or between C1 and C2. Labeled spinocerebellar tract neurons were classified into two groups according to the axonal course in the spinal cord; one is composed of neurons with uncrossed ascending axons and the other, neurons with crossed ascending axons. Neurons of origin of the uncrossed tracts were located in the medial part of lamina VI of C2 to C8, the central part of lamina VII of C4 to C8, lamina V of C7 to L3 and Clarkes column. Neurons of origin of the crossed tracts were found in the central cervical nucleus of C1 to C3, the intermediate zone and the ventral horn of the lower thoracic and the lumbar segments (T11 to L3), and in the dorsal horn, the medial part of lamina VII and the ventrolateral part of the ventral horn of the sacral and caudal spinal cord. In comparison with our previous results in the cat, it was suggested that the spinocerebellar system in the rat is organized in the same fashion as in the cat, in terms of the location and the intraspinal axonal course of the cells of origin.

The distribution of spinal projection neurons in the hypothalamus of the rat, studied with the HRP method.

SummaryThe distribution and number of hypothalamospinal tract (HST) neurons were studied following injections of horseradish peroxidase (HRP) at various levels of the rat spinal cord. The hypothalamus was divided into four areas and one nucleus, that is, the dorsal (DHA), posterior (PHA), medial (MHA) and lateral (LHA) hypothalamic areas and the paraventricular nucleus (PVN).The total numbers of HST neurons labeled with HRP varied according to the injection levels: 6,160 (C2 injections), 3,808 (T8), 1,961 (L1), 919 (L7) and 13 (S4). With C2 injections LHA contained 3,464 neurons, which accounted for 56% of the full number of HST neurons; similarly, PVN, 1,114 (18%); MHA, 865 (14%); DHA and PHA, 817 (12%). With L7 injections, LHA contained 444 labeled neurons, which accounted for 48% of the total; PVN, 327 (36%); MHA, 71 (8%); DHA with PHA, 77 (8%). As for the rostrocaudal distribution of labeled neurons, there was only a slight difference between the C2 and L6 injections in LHA, but no difference was noticed in PVN, DHA nor PHA.The present findings suggest that 70% of HST neurons may project to the cervical and thoracic cords. Although the number of labeled HST neurons decreased as the injection sites were placed caudally, no clearcut topographical arrangement was recognized in terms of the spinal projection levels.

A direct hypothalamic projection to the superior salivatory nucleus neurons in the rat. A study using anterograde autoradiographic and retrograde HRP methods

The location of the superior salivatory nucleus and terminal labelings of the hypothalamic descending fibers were demonstrated in the nucleus reticularis parvocellularis using HRP and the autoradiographic techniques, respectively. When both techniques were used in the same animals, some HRP-labeled neurons were seen among the accumulations of silver grains, suggesting pericellular terminations. The present study demonstrates that the hypothalamic efferents project directly to the superior salivatory nucleus innervating salivary and lacrimal glands.

Hypothalamic projections to the ventral medulla oblongata in the rat, with special reference to the nucleus raphe pallidus: a study using autoradiographic and HRP techniques

Hypothalamic descending projections to the medullary ventral surface were studied autoradiographically in the rat. A small amount of [3H]leucine was injected unilaterally into various parts of the hypothalamus by air pressure. Abundant and characteristic terminal labelings were observed bilaterally in the nucleus raphe pallidus, the ventral surface of the pyramidal tract and the nucleus interfascicularis hypoglossi, after injections into the dorsal posterior hypothalamic area caudal to the paraventricular hypothalamic nucleus. Conspicuous, but less numerous labelings were observed in the nucleus raphe obscurus and the ipsilateral raphe magnus. After an injection of [3H]leucine into the hypothalamus and injections of horseradish peroxidase (HRP) into the spinal cord in the same animal, silver grains were densely distributed around HRP-labeled neurons in the nucleus raphe pallidus including the nucleus interfascicularis hypoglossi. The present results suggest that the dorsal posterior hypothalamic area projects directly to the spinal-projecting neurons of the nucleus raphe pallidus.

A direct projection from the hypothalamus to the area postrema in the rat, as demonstrated by the HRP and autoradiographic methods

A direct hypothalamic afferent to the area postrema was found in the rat. After an injection of tritiated amino acids into the hypothalamus, labeled fibers and terminals were observed in the entire part of the area postrema, with dense patches of silver grains present in places. Cells of origin of this novel projection were identified with the retrograde horseradish peroxidase (HRP) method in the dorsal hypothalamic area at the most rostral level of the dorsomedial hypothalamic nucleus.

The location of spinal projection neurons in the cerebellar nuclei (cerebellospinal tract neurons) of the cat. A study with the horseradish peroxidase technique.

The distribution of spinal projection neurons was studied in the cerebellar nuclei of the cat following injections of horseradish peroxidase (HRP) into the cervical, thoracic and lumbar cord. HRP-positive (labeled) neurons were found in the medial (fastigial) and the posterior interpositus nuclei on the side contralateral to the cervical injection, being most numberous in cases with injections between the C2 and the C3 segments. In the medial nucleus (M) labeled neurons were distributed in the central to the caudal portions, and there was a conspicuous group of labeled small neurons extending from the ventrolateral part to the intermediate zone between the M and the anterior interpositus nucleus. With an increasing number of medium-sized neurons, this neuronal group persisted caudally in a similar position, ventromedial to the posterior interpositus nucleus (IP). Labeled large neurons were seen in the medial third of the IP. In the two cases labeled neurons of medium and small sizes were equal in number, and the neurons of the IP constituted about 10% of the total number of the spinal projection neurons. The present study suggests that the neurons of the M and the IP, including those of the intermediate group located between the two, project the bulk of the crossed descending fibers as far caudally as the C2 and the C3 segments.