Toshiko Kuchiiwa

Morphological study of orexin neurons in the hypothalamus of the Long-Evans rat, with special reference to co-expression of orexin and NADPH-diaphorase or nitric oxide synthase activities.

Orexins, novel neuropeptides, are exclusively localized in the hypothalamus and implicated in the regulation of a variety of activities, including food intake and energy balance. Nitric oxide (NO), an unconventional neurotransmitter, is widely present in numerous brain regions including the hypothalamus, and has similar physiological roles to those of the orexins. The present study was undertaken to examine the distribution of orexin neurons and the presence of neuronal nitric oxide synthase (nNOS) in the orexin neurons to clarify whether NO interacts with the orexins in the neuronal regulation activities in the Long-Evans rat. We used two double-labeling methods: nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry in combination with orexin immunohistochemistry, and double-labeling fluorescent immunohistochemistry for orexin and nNOS. The majority of the orexin immunoreactive neurons were localized mainly in the areas of the dorsomedial hypothalamic nucleus (DMN), the dorsal part of the perifornical nucleus (PEF) and lateral hypothalamic area. The orexin immunoreactive cell bodies were medium in size, and triangular, round, elliptic, and fusiform in shape. The sizes and shapes of orexin neurons in the different parts were similar. Cell bodies coexpressing the orexin and nNOS or NADPH-d were present in the areas of the DMN and the PEF, and the nerve fibers containing orexin and nNOS were distributed in the DMN and PEF, arcuate nucleus (ARN) and ventromedial hypothalamic nucleus (VMH). These results provide morphological evidence that there exists a population of nNOS- or NADPH-d-/orexin-coexpressing neurons in the orexinergic cell group in the hypothalamus, and taken together with previous findings, suggest that NO may play a role in the mechanisms by which orexin neurons regulate food intake and energy balance.

Nadph diaphorase neurones are evenly distributed throughout cat neocortex irrespective of functional specialization of each region

Using NADPH diaphorase histochemistry as a marker for nitric oxide synthase we investigated the possible sites of nitric oxide synthesis in cat cerebral neocortex. Intensely stained neurones were found mainly in the deep layers of the neocortex and underlying medulla. Virtually all neurones in the cerebral medulla were NADPH diaphorase positive. The density of diaphorase neurones was estimated in the cortex/medulla border zones of each neocortical gyrus. Diaphorase neurones were evenly distributed throughout the neocortex and no significant statistical difference between gyri was observed. These findings indicate that the density of diaphorase neurones is irrespective of functional specialization of each region and are more in line with the hypothesis that NADPH diaphorase neurones are involved in the control of local cortical blood flow.

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin decreases serotonin-immunoreactive neurons in raphe nuclei of male mouse offspring

Female ddY mice were administered 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by gavage for 8 weeks prior to pregnancy. In the male breast-fed offspring born to the TCDD-exposed mice, serotonergic neurons in the brainstem were examined using an immunocytochemical method at 42 days of age. In all offspring, a marked decrease in the intensity of immunostaining occurred in all raphe nuclei compared with the control offspring. The number of serotonin-immunoreactive neurons in each raphe nucleus was measured by computer-assisted analysis. Approximately a quarter to half of immunoreactive neurons were detected in the TCDD-exposed offspring raphe nuclei compared with the control offspring. The present findings suggest that in utero and/or lactational TCDD exposure cause a long-lasting change in the serotonergic system in the raphe nuclei of offspring.

Presence of neuronal nitric oxide synthase in autonomic and sensory ganglion neurons innervating the lacrimal glands of the cat: an immunofluorescent and retrograde tracer double-labeling study.

It is generally considered that parasympathetic postganglionic nerve fibers innervating the lacrimal gland (LG) arise from the pterygopalatine ganglion (PPG), while sympathetic and sensory innervations arise from the superior cervical ganglion (SCG) and trigeminal ganglion (TG), respectively. Recently, we reported for the first time that the parasympathetic innervation of the cat LG was also provided by the otic ganglion (OG) and ciliary ganglion (CG), and that the sensory innervation was also provided by the superior vagal ganglion (SVG) and superior glossopharyngeal ganglion (SGG). To determine if nitric oxide (NO) is a neurotransmitter of the autonomic and sensory neurons innervating the LG, we injected the cholera toxin B subunit (CTB) as a retrograde tracer into the cat LG, and used double-labeling fluorescent immunohistochemistry for CTB and nitric oxide synthase (NOS). We found that NOS-/CTB-immunofluorescent double-labeled perikarya were localized in the PPG, OG, TG, SVG and SGG, but not in the CG and SCG. The highest numbers of NOS-/CTB-immunofluorescent double-labeled neurons were found in the PPG and TG. In addition, we examined the presence of nitrergic nerve fibers in the LG using NADPH-d histochemistry and found that a large amount of NADPH-d-stained nerve fibers were distributed around the glandular acini and in the walls of glandular ducts and blood vessels. This study provides the first direct evidence showing that NO may act as a neurotransmitter or modulator involved in the parasympathetic and sensory regulation of lacrimal secretion and blood circulation, but may not be implicated in the sympathetic control of LG activities, and that nitrergic nerve fibers in the LG arise mainly from parasympathetic postganglionic neurons in the PPG and sensory neurons in the TG. The present results suggest that NO plays an important role in the regulation of LG activities.

Morphological distinction between vasodilator and secretomotor neurons in the pterygopalatine ganglion of the cat

We investigated whether vasodilator and secretomotor ganglion neurons are morphologically distinguishable from each other in the parasympathetic ganglion of the cat. When Cholera toxin B subunit, a retrograde tracer, was injected into the palatine gland, both large and small ganglion neurons were retrogradely labeled in the pterygopalatine ganglion. On the other hand, when the tracer was injected into gland-free areas (the upper gingiva or epidural space), all neurons labeled in the ganglion were small in size. Thus, it was assumed that small and large neurons labeled in the ganglion represented, respectively, secretomotor and vasomotor neurons.

Autonomic and sensory innervation of cat molar gland and blood vessels in the lower lip, gingiva and cheek.

Innervation of the molar gland and blood vessels in the lower lip, gingiva and cheek mucous membrane was investigated in the cat with the aid of whole mount acetylthiocholinesterase (WATChE) histochemistry and retrograde neuronal tracing methods with horseradish peroxidase (HRP) and HRP-conjugated wheat germ agglutinin (WGA-HRP). The molar gland was found to be supplied from the buccal nerve and branches of the mylohyoid nerve on the basis of microdissection of WATChE-stained mandibular preparations under a dissecting microscope. The rostral half of the lower lip-gingiva was innervated by mental branches from the inferior alveolar nerve. The caudal half of the lower lip-gingiva and cheek mucous membrane were observed to be supplied from the buccal nerve. Following injections of HRP/WGA-HRP into the molar gland, lower lip-gingiva and cheek, many retrogradely labeled ganglion neurons were observed in the ipsilateral main and accessory otic ganglia, superior cervical ganglion and mandibular division of the trigeminal ganglion. In the pterygopalatine ganglion, a small number of positive neurons were found, but in a few cases in which the injected tracer was restricted to the lower lip-gingiva and anterior half of the molar gland, labeled neurons were not detected in the main ganglion nor in its accessory microganglia. These findings indicate that the cat molar gland receives a postganglionic parasympathetic supply from the otic ganglia, postganglionic sympathetic input from the superior cervical ganglion and sensory innervation from the trigeminal ganglion by way of the buccal nerve and mylohyoid nerve. Vessels in the rostral half of the lower lip-gingiva receive the same inputs from the inferior alveolar nerve, and vessels in the caudal half receive inputs from the buccal nerve. The vessels in the cheek mucous membrane receive dual parasympathetic supplies from the otic ganglia and the pterygopalatine ganglion by way of the buccal nerve.

Three novel neural pathways to the lacrimal glands of the cat: an investigation with cholera toxin B subunit as a retrograde tracer.

The distribution of ganglion neurons innervating the lacrimal gland (LG) was investigated following injection of cholera toxin B subunit into the LG of the cat. We report the first evidence that the otic ganglion (OG), and superior vagal and glossopharyngeal ganglia are also the sources of innervation of the LG. LG-innervating neurons in the pterygopalatine ganglion and the OG could be divided into two subpopulations: small and large neurons. They may mediate the vasodilatation and secretion, respectively.

Dioxin exposure down-regulates nitric oxide synthase and NADPH-diaphorase activities in the hypothalamus of Long-Evans rat

In this study, we investigated the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) gastric administration on the expression of neuronal nitric oxide synthase (nNOS) and the NADPH-diaphorase (NADPH-d) activities in the brain of the Long-Evans rat. A single dose of TCDD (dissolved in olive oil, 50 microg/kg) or olive oil alone was administered to the rats by gavage. nNOS Western blotting experiment indicated a marked decrease in nNOS immunoreactivity at 1 and 2 weeks after TCDD treatment. NADPH-d histochemistry results showed a marked decrease in the number of NADPH-d stained cell bodies in the paraventricular hypothalamic nucleus, lateral hypothalamic area and perifornical nucleus in the TCDD-treated rats. The present study suggests that TCDD administration down-regulates nitric oxide product in the hypothalamus, which may be partially responsible for TCDD-induced feeding inhibition.

Up-regulation of methionine-enkephalin-like immunoreactivity by 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment in the forebrain of the Long-Evans rat.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is considered to be one of the most toxic environmental contaminants, named dioxin. Exposure to TCDD induces a plethora of intoxication symptoms, including anorexia and hypothermia, in several mammals and human. Enkephalin, an endogenous pentapeptide, is an important neuroregulator of autonomic functions, such as food intake and body temperature. In this study, we investigated the effects of TCDD gastric administration on methionine-enkephalin (MEK) immunoreactivity in the brain of the Long-Evans rat, the species strain considered to be the most TCDD-susceptible, using immunohistochemical staining. A single dose of TCDD (dissolved in olive oil, 50 microg/kg) or olive oil alone was administrated to the rats by gavage. Compared with the vehicle-treated rat, a marked increase in the density of MEK immunoreactive cell bodies, fibers and terminals was found 2 weeks after TCDD treatment in the forebrain of the TCDD-treated rat, i.e. the central amygdaloid nucleus, field CA3 of the hippocampus, paraventricular hypothalamic nucleus, medial preoptic nucleus, interstitial nucleus of the posterior limb of the anterior commissure, lateral globus pallidus, ventral pallidum and lateral division of the bed nucleus of the stria terminalis. These results demonstrated for the first time a site-specific increased enkephalinergic activity in certain brain regions of the Long-Evans rat. It is suggested that the increased MEK immunoreactivity may act as a compensatory adaptation for the pathophysiological alterations caused by TCDD exposure.

Facial nerve parasympathetic preganglionic afferents to the accessory otic ganglia by way of the chorda tympani nerve in the cat

Abstract The distribution of accessory otic ganglia and connections between the ganglia and the chorda tympani nerve were investigated in the cat in order to determine the parasympathetic preganglionic facial nerve afferents to the otic ganglia using whole mount acetylthiocholinesterase (WATChE) histochemistry. The otic ganglia consist of a sigle main prominent ganglion and many small accessory ganglia lying on a plexus around the origins of the branches of the mandibular nerve and near the junction of the chorda tympani nerve and lingual nerve. In cell analysis of Nissl-stained preparations, the neurons composing the accessory otic ganglia were morphologically similar to the main otic ganglion neurons. Connecting branches from the chorda tympani nerve to the peripherally located acccessory otic ganglia were found and they were not stained by WATChE histochemistry. WATChE-positive connecting branches from the ganglia to the inferior alveolar, lingual, and mylohyoid nerves were also found in the same preparations. The WATChE histochemistry on various autonomic nervous tissues revealed that autonomic postganglionic nerve fibers are selectively stained darkly and that preganglionic fibers remain unstained. Therefore, it is considered that the WATChE-negative connections from the chordra tympani nerve consist chiefly of autonomic preganglionic fibers, whereas the WATChE-positive connections to the branches of the mandibular nerve are mainly postganglionic fibers. This suggests that some of the facial nerve parasympathetic preganglionic fibers in the chorda tympani nerve are mediated in the accessory otic ganglia and then join the branches of the mandibular nerve to supply the target mandibular tissues.