Norihiko Ihara

Distribution of the endogenous digitalis-like substance (EDLS)-containing neurons labeled by digoxin antibody in hypothalamus and three circumventricular organs of dog and macaque

Endogenous digitalis-like substance (EDLS) is a newly discovered humoral agent which causes sodium-diuresis. EDLS is well known to have inhibitory activity to Na+,K(+)-ATPase and cross-immunoreactivity to digoxin antibody; however, its precise chemical structure has not yet been determined. We had previously developed a histochemical technique for EDLS, i.e., digoxin-immunohistochemistry, and demonstrated that EDLS was produced in the hypothalamic neurons. In the present study, the distribution of EDLS-containing neurons in the hypothalamus of dog and macaque was investigated using this technique, because anti-EDLS antibody cannot be obtained yet. In both species, EDLS neuronal somata were mainly localized in the paraventricular nucleus and the supraoptic nucleus and its accessory nuclei. A number of somata were also scattered in the other hypothalamic areas. The processes of these neurons ran from the area where the somata were located, through the lateral and basal area of the hypothalamus, to the infundibulum. These nerve fibers with varicosities were associated with the primary capillaries of hypophysial portal veins. A few immunopositive nerve fibers were also seen in the pituitary posterior lobe of both species. Intensive immunoreactivities were observed in the subfornical organ and organum vasculosum laminae terminalis. There were no differences between dog and macaque.

Effects of lansoprazole on pharmacokinetics and metabolism of theophylline

The effect of the new substituted benzimidazole proton pump inhibitor, lansoprazole, on pharmacokinetics and metabolism of theophylline has been studied in healthy adults given oral lansoprazole 30 mg once daily for 11 days. On Days 4 and 11 of 300 mg aminophylline was simultaneously administered orally and blood samples for theophylline analysis were taken over 24 h. Urine samples were collected for up to 24 h and were assayed for theophylline and its major metabolites 1,3-dimethyluric acid (1,3-DMU), 1-methyluric acid (1-MU) and 3-methylxanthine (3-MX). The pharmacokinetic parameters of theophylline were determined, and the urinary recovery of unchanged theophylline and its major metabolites were calculated.After administration of lansoprazole for 4 days, no significant alteration in the terminal elimination half-life (t1/2β) or the mean residence time (MRT) was detected. However, there was a significant decrease of about 13% in the area under the plasma concentration-time curve (AUC) and a significant increase of about 19% in the apparent clearance (CLapp). Lansoprazole treatment for 11 days caused a significant decrease of approximately 12% in t1/2β and about 10% in the MRT of theophylline, although neither AUC nor CLapp showed a significant alteration. The excretion of 3-MX in the urine was significantly increased by about 20% after lansoprazole treatment for 4 and 11 days, although there was no significant alteration in the excretion of unchanged theophylline, 1,3-DMU or 1-MU.The results indicate that repeated administration of lansoprazole to humans induces the hepatic microsomal P-450-dependent drug oxidation system that mediates N-1-demethylation of theophylline, consequently increasing its metabolism.

Reinnervation of serotonin fibers in the denervated rat subcommissural organ by fetal raphe transplants. An immunohistochemical study.

The ability of axonal outgrowth of serotonin neurons in the implanted brain tissue of subcommissural organ (SCO) was immunohistochemically studied. The serotonin neuron system of the experimental rats was completely destroyed by the intraventricular injection of 5,6-dihydroxytryptamine. The raphe region of normal fetal rats was implanted into the caudal part of the third ventricle of the neurotoxic drug pretreated rats. The host brain was examined 3 months after transplantation. The numerous serotonin fibers were distributed in the SCO and periventricular region of the third ventricle of the host brain. The outgrowing serotonin fibers from the raphe transplant seemed to innervate the SCO with the target specificity.

The organization of serotonin fibers in the mammalian superior colliculus. An immunohistochemical study.

SummaryThe distribution of serotonin immunoreactivity in the superior colliculus (SC) of the rat, hamster, chipmunk, cat, and monkey was studied using a sensitive immunohistochemical method. In all of these animals, serotonin immunoreactivity formed a dense network of varicose fibers throughout the SC. These fibers had a characteristic arrangement corresponding to the laminar structures of the SC. Except in the chipmunk, serotonergic fibers were more dense in the stratum griseum superficiale than in the other layers. In the SC of the chipmunk, these fibers appeared evenly distributed.To explore the degree of scrotonergic innervation in each layer, a semi-quantitative assay of serotonin immunoreactive varicosities was conducted in the rat, chipmunk, cat, and monkey. Peaks in varicose density were seen in the stratum griseum superficiale, the stratum griseum intermedium and the stratum griseum profundum. In the rat, cat, and monkey, the highest density of these varicosities was in the stratum griseum superficiale. On the other hand, the stratum griseum intermedium of the chipmunk SC received the greatest innervation of serotonergic varicose fibers.

Immunohistochemical demonstration of serotonin-containing nerve fibers in the mammalian hippocampal formation

The distributional pattern of serotonin-immunoreactive nerve fibers in the hippocampal formation of six different mammalian species (rat, chipmunk, hamster, cat, dog and monkey) was studied in detail by means of a modified peroxidase-antiperoxidase immunohistochemical method, using a specific serotonin antiserum. Furthermore, the density of varicosities distributed in each layer of the hippocampus was semiquantitatively analyzed. In all species investigated, the routes of serotonin fibers entering the hippocampal formation were found to be almost the same. These fibers were extensively distributed throughout the hippocampal formation, and had a characteristic arrangement corresponding to the laminar structure of this region. A dense innervation by varicose serotonin fibers was found in the stratum lacunosum-moleculare, but a few serotonin fibers were also distributed in the stratum lucidum of the CA2 and CA3 fields. The stratum pyramidale and the granule cell layer of the dentate gyrus contained a small number of serotonin fibers. The concentration and the direction of serotonin fibers were different in each area of each animal. Two peculiar observations should be stressed: (1) in the rat, the strata oriens and radiatum of CA2 and CA3 as well as the stratum lacunosum-moleculare displayed abundant serotonin fibers; (2) in the dog, abundant serotonin fibers were diffusely distributed in the CA1 field except for the stratum pyramidale and the most dense concentration of serotonin fibers was seen in the stratum oriens of CA3. The present study provides a morphological basis for further study of the functional significance of serotonin in the limbic system.

Rearrangement of serotonin-immunoreactive fibers in the denervated rat suprachiasmatic nucleus after transplantation of fetal raphe tissue.

SummaryPieces of fetal midbrain raphe tissue were transplanted into the third ventricle or the ventral hypothalamic region near the suprachiasmatic nucleus (SCN) of adult host rats that had previously been denervated by treatment with 5,6-dihydroxytryptamine. The ability of grafted serotonin neurons to reinnervate the SCN in the host rats was studied by means of immunohistochemistry 1 and 3 months after transplantation. In both the intraventricular and intraparenchymal transplant experiments, reinnervation by outgrowing serotonin fibers was observed in the hypothalamus of host rats at 1 and 3 months after surgery. At both survival periods, there was no abundant arborization of serotonin fibers in the SCN, while the preoptic and periventricular areas of the host rats displayed a pattern of serotonergic innervation resembling that in normal (untreated) rats. It is suggested that within the SCN the regenerating serotonin fibers may be exposed to an inhibitory environment.

Ouabain-like immunoreactive substances exist in the hypothalamus and the adrenal medulla in rats

Abstract Since it has been demonstrated that one of the endogenous digitalis-like factors (EDLF) is ouabain, searches for the EDLF are focused on the endogenous ouabain in the mammalian body. We have developed a monoclonal, anti-ouabain antibody which similarly cross-immunoreacts with other digitalis glycosides such as ouabain, digoxin, and digitoxin; the epitope of this antibody was confirmed to be the partial structure important for cardiac compounds and neutralizing their pharmacological activity, Na + /K + -ATPase inhibition, in a competitive mode against K + . By using this monoclonal antibody, we immunohistochemically examined the distribution of the digitalis-like substances in the adrenal glands and the hypothalamus. The immunoreactivity was diffusely distributed in the cytoplasm of endocrine cells of the adrenal medulla, but not in the adrenal cortex. Immunoreactive neuronal somata and their proximal processes were found in the hypothalamic paraventricular nucleus, and the intensity of the immunostaining in the proximal processes was greater than in the somata. In the neurons of the supraoptic nucleus very weak immunoreactivity was observed. No immunoreactive material was detected in the other areas of the hypothalamus examined in this study. Present findings with a monoclonal, ouabain antibody suggest that the ouabain-like immunoreactive substances are exclusively distributed in the neural tissues such as the hypothalamus and the adrenal medulla. Although the physiological role of the substance is still uncertain, this particular distribution suggests that it may be a neurotransmitter implicated in the cardiovascular and endocrine functions.

Immunohistochemical study on fetal raphe samples transplanted into the leptomeningeal tissue of 5,6-dihydroxytryptamine-treated adult rats.

SummaryPieces of fetal midbrain raphe containing serotonergic and dopaminergic neurons were transplanted into the leptomeningeal tissue (see Fig. 3) of adult host rats that had previously been denervated by treatment with 5,6-dihydroxytryptamine. One, 2 and 5 months after transplantation, the rate of neuronal survival in the grafted tissue and the extent of axonal outgrowth into the host brain were studied by use of serotonin and tyrosine hydroxylase (TH) immunohistochemistry. The survival rate of the grafts in the 1-month group was approximately 70%. Neurons containing either serotonin or catecholamine were demonstrated by means of immunocytochemical procedures in the grafts. Two and 5 months after transplantation, serotonin-immunoreactive nerve fibers were densely distributed throughout the graft tissue, while TH-immunoreactive fiber elements were restricted to an area near the somata of TH-positive neurons. Numerous serotonin-immunoreactive fibers derived from the transplant were found in the leptomeningeal tissue surrounding the graft, on the wall of neighboring blood vessels, and also in the adjacent parenchyma of the host brain. Outgrowing TH-immunoreactive nerve fibers were not observed in the host brain, although such elements occurred in the leptomeningeal tissue and the wall of the larger blood vessels. These results suggest that the serotonergic and catecholaminergic (dopaminergic) neurons located in transplants of the raphe nuclei show different patterns when reinnervating the host tissue.

Transplantation of fetal mesencephalic and medullary raphe tissues into the cerebellum of denervated adult rats — an immunohitochemical study

Pieces of mesencephalic and medullary raphe tissues were transplanted into the cerebella of 5,6-dihydroxytryptamine-treated adult rats. The extent of axonal outgrowth of serotonergic and dopaminergic neurons in the grafts was immunohistochemically studied. At 3 months after transplantation, numerous dopaminergic neurons with many processes extending within the graft were detected in the mesencephalic raphe graft, but not in the medullary raphe graft. In contrast, both the mesencephalic and medullary raphe grafts contained numerous serotonergic neurons and a dense plexus of their fibers. The outgrowing serotonergic fibers from the mesencephalic raphe graft showed a hyperinnervation pattern in the cerebellar cortex adjacent to the graft. Furthermore, a glomerulus-like accumulation of serotonergic fibers was observed in the granular layer. In the cases of medullary raphe grafts, the relatively abundant outgrowing serotonergic fibers showed a laminar organization in the cerebellar cortex near the graft, which was similar to the normal distributional pattern. These results indicate that serotonergic and dopaminergic neurons located within the mesencephalic raphe graft clearly differed from each other in their ability to extend their processes into the host cerebellum, which provides further evidence for the existence of specific interactions between outgrowing serotonergic fibers and their terminal fields (targets).

Immunohistochemical investigations of the influence of reserpine on the serotonin neuron system in the rat brain

A peroxidase-antiperoxidase immunohistochemical method with serotonin antiserum was employed to investigate the influence of reserpine on serotonin neurons of rats which were sacrificed at various times after injection (10 mg/kg i.p.). The disappearance of serotonin immunoreactivity induced by reserpine was detected only in the perikarya after 15 min, and then rapidly proceeded to the terminals. Between 2 and 4 h, immunoreactivity completely disappeared throughout the brain. The immunoreactivity reappeared in the perikarya after 6 h, and progressed toward the terminals gradually. However, there was an obvious difference in the rate of recovery of immunoreactivity between areas. After 7 days, the immunoreactivity returned to control levels.