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.

Age-related alterations of nitric oxide production in the brains of seizure-susceptible EL mice.

We evaluated age-related changes in nitric oxide (NO) production in the brains of EL mice, a strain highly susceptible to seizures. A group of EL(s) mice were tossed up weekly to induce convulsive seizures, while in a nonstimulated EL(ns) group induction of convulsive seizures was avoided. Brain levels of nitrite plus nitrate (NOx) in EL(ns) mice were significantly higher than in nonstimulated mice at 10 days, and also higher than levels at 15 and 50 weeks in either EL(s) or EL(ns) mice. A significantly higher number of NO-producing cells were demonstrated in the hippocampus and parietal cortex by staining for nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase in EL(s) mice at the ages of 15 and 50 weeks than in EL(ns) mice at the age of 6 weeks. In EL(ns) mice, significantly fewer neurons showed NADPH-diaphorase staining in the hippocampus, striatum and parietal cortex at the age of 50 weeks than at 6 weeks. The present results suggest that whole-brain NOx levels in EL(ns) and EL(s) mice and numbers of NADPH-diaphorase-positive neurons in EL(ns) mice decreased with aging, while increasing of numbers of such neurons in EL(s) mice were assumed to develop in compensation for reduction in whole-brain NOx levels.

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.

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, vasomotor and secretomotor and neurons [corrected].

The effect of alcohol on convulsions and nitric oxide levels in seizure-susceptible el mice.

To evaluate the influences of ethanol intake on convulsive seizures and brain nitric oxide (NO) production, EL mice, a strain highly susceptible to seizures, were given a 10% ethanol solution ad libitum. In mice consuming ethanol for 4, 8, and 12 weeks, seizures were not suppressed by zonisamide (75 mg/kg ip). Brain NO metabolite levels in mice after 12 weeks of consumption were significantly lower than those in control mice and those consuming ethanol for 4 weeks. Numbers of NADPH diaphorase-positive neurons in the hippocampal formation and parietal cortex of mice consuming for 4 and 12 weeks were significantly higher than in controls. These results suggested that increasing of numbers of NADPH diaphorase-positive neurons in the hippocampal formation and parietal cortex were assumed to develop in compensation for reduction in whole-brain NO metabolite levels of EL mice exposed to ethanol.

Nitric oxide production is decreased in the brain of the seizure susceptible EL mouse.

To investigate nitric oxide production in the brain of the EL mouse, an inbred mutant strain of the ddY mouse that is susceptible to convulsive seizures, we measured whole brain nitric oxide metabolites, and counted the number of nitric oxide-producing cells in the parietal cortex and striatum. Nitric oxide metabolites in the brain and serum were determined by measuring levels of nitrite plus nitrate. Nitric oxide-producing cells were demonstrated histochemically by staining for nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase. Levels of nitrite plus nitrate in the whole brain were significantly lower than those of the control mice, although levels of nitrite plus nitrate in the serum did not differ between groups. There were significantly fewer NADPH-diaphorase-positive cells in the parietal cortex and striatum of the EL mouse compared to the ddY controls. These results suggest that lower nitric oxide production in the brain may be related to the susceptibility of the EL mouse to convulsive seizures.

Evidence for the collateral innervation of the esophagus and the heart from neurons in the compact formation of the nucleus ambiguus of the rat.

We investigated whether the heart receives collateral projections from the neurons which innervate the esophagus with a retrograde double-labeling method using two fluorescent tracers. Following injections of True Blue (TB) into the esophagus and Diamidino Yellow (DY) into the heart, about 21.9% of the labeled esophageal motoneurons in the compact formation of the nucleus ambiguus (AmC) were retrogradely double labeled. No single-labeled cardiac motoneurons were found in the AmC. The present results provide anatomical substrates for the esophagocardiac reflex.

Kainic and domoic acids differentially affect NADPH-diaphorase neurons in the mouse hippocampal formation.

We investigated changes in numbers of nitric-oxide-producing cells in the hippocampal formation, striatum, and temporal cortex of mice 24 h after intraperitoneal administration of kainic acid (5, 10, 15, and 20 mg/kg) or domoic acid (1, 2, and 4 mg/kg). Nitric-oxide-producing cells were demonstrated histochemically by staining for nicotinamide adenine dinucleotide phosphate diaphorase. Nicotinamide adenine dinucleotide phosphate-diaphorase-positive neurons in the dentate gyrus and the subiculum did not change in number following administration of kainic acid or domoic acid at any dose. Positive neurons in the CA3 region of mice treated with kainic acid or domoic acid at any dose were significantly fewer than in controls. Although the numbers of positive neurons in the CA1/CA2 regions did not differ from those of controls at any of the four doses of kainic acid, positive cells in the CA1/CA2 were significantly more numerous than in controls at any dose of domoic acid. Although no significant differences in the numbers of positive neurons in the striatum were apparent between controls and any of the four doses of kainic acid, domoic acid significantly decreased the numbers of such cells. These results suggest that systemically administered kainic acid and domoic acid affect differentially nitric-oxide-producing cells in the hippocampal formation.

The retinal projections to the ventral and dorsal divisions of the medial terminal nucleus and mesencephalic reticular formation in the Japanese monkey (Macaca fuscata): a reinvestigation with cholera toxin B subunit as an anterograde tracer.

After a monocular injection of the cholera toxin B subunit (CTB) into the vitreous chamber of the eye, retinal projections to the medial terminal nucleus (MTN) of the accessory optic system (AOS) were studied in the Japanese monkey. The anterogradely transported tracer was visualized with the peroxidase antibody technique by using an anti-cholera toxin antibody. One small accumulation of the CTB-immunopositive retinofugal terminals was located in a small area just medial to the medial edge of the cerebral peduncle and anterior to the attachment of the oculomotor nerve, suggesting the existence of a ventral division of the MTN of the AOS. Caudally, one very small bundle of the retinofugal fibers extending dorsally from this accumulation was seen running along the medial edge of the cerebral peduncle and substantia nigra to the small region corresponding to the dorsal division of the MTN. A few small bundles of CTB-immunopositive retinal fibers were observed to leave the superior fasciculus of the AOS at various points. These fibers coursed medially through the cerebral peduncle and substantia nigra to reach some restricted areas of the mesencephalic reticular formation between the medial lemniscus and the substantia nigra.

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

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 single 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 accessory 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 chorda 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.

Influence of dietary zinc on convulsive seizures and hippocampal NADPH diaphorase-positive neurons in seizure susceptible EL mouse.

Adequate, high and deficient dietary levels of zinc (Zn) were compared in seizure-susceptible EL mice with respect to convulsions and to nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase-positive hippocampal neurons. Diaphorase positivity is associated with nitric oxide (NO) production. Convulsive seizures in the EL mice given the various diets did not differ over 1-4 weeks, but convulsions in EL mice given the Zn-deficient diet for 4 weeks were more effectively suppressed by injection of zonisamide (ZNS) (75 mg/kg intraperitoneally) than in mice receiving high- or adequate-Zn diet for the same period. Numbers of NADPH diaphorase-positive neurons in the CA1/CA2 region of the hippocampal formation were significantly higher in mice given the Zn-deficient diet for 4 weeks than in mice fed adequate Zn. Mice receiving the high-Zn diet for the same period had significantly fewer NADPH diaphorase-positive neurons in the subiculum than mice with adequate Zn. These results suggest that Zn deficiency inhibits convulsive seizures of EL mice, and that dietary Zn influences numbers of NO producing neurons in the hippocampal formation.

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.

Sparse distribution of NADPH diaphorase neurons in the hippocampal formation of the inbred mutant strain EL mouse.

The number of NADPH diaphorase-positive cells in the CA1/CA2 and CA3 regions of Ammon's horn and the subiculum of the hippocampal formation of EL mice, an inbred mutant strain of the ddY mouse susceptible to convulsive seizures, was fewer than that of ddY mice. These findings suggest that smaller numbers of nitric oxide producing cells in the hippocampal formations of EL mice is related to their susceptibility to convulsive seizures.

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.

Localization of preganglionic neurons of the accessory ciliary ganglion in the midbrain: HRP and WGA-HRP studies in the cat.

Localization of preganglionic neurons of the accessory ciliary ganglion (ACG), including ectopic intraocular ganglion cells, was investigated in the cat with the aid of horseradish peroxidase (HRP) and HRP-conjugated wheat germ agglutinin (WGA-HRP) methods. When HRP or WGA-HRP was injected into the anterior and posterior chambers of the eye, no retrogradely labeled cells were found in the visceral oculomotor nuclei, although most neurons of the ACG and the main ciliary ganglion (CG) were intensely labeled. When a microsyringe needle was inserted into the ciliary body, the tracer diffused into the suprachoroid lamina and the intraocular ganglion cells, and a small number of labeled neurons appeared in the midplane between each side of the somatic oculomotor nuclei. After injection into the ACG, many labeled neurons were observed in the anteromedian nucleus, Edinger-Westphal nucleus, and midplane between the somatic oculomotor nuclei, their ventral continuations of the ventral tegmental area, and the periaqueductal gray. HRP/WGA-HRP injection into the CG labeled cells in all these areas and in the lateral border zones of the anteromedian, Edinger-Westphal and somatic oculomotor nuclei, and their ventral continuations of the ventral tegmental area. These findings indicate that the visceral oculomotor neurons which project to the ACG tend to be located more medially than those to the CG.

Oculomotor parasympathetic pathway to the accessory ciliary ganglion bypassing the main ciliary ganglion by way of the trigeminal nerve.

When an HRP or WGA-HRP solution was injected into the rostral midbrain including the oculomotor visceral nuclei, densely distributed HRP/WGA-HRP-positive granules were observed around the ganglion neurons in the accessory ciliary ganglion (ACG) and ectopic neurons in the communicating branch from the long ciliary nerve to the ACG. The same injections labeled fibers within the communicating branch as well as the fibers between the ACG and the main ciliary ganglion (CG). These findings indicate that some oculomotor parasympathetic preganglionic fibers reach the ACG bypassing the CG by way of the trigeminal nerve.

Origins of parasympathetic postganglionic vasodilator fibers supplying the lips and gingivae; an WGA-HRP study in the cat.

Application of WGA-HRP to the mandibular lip and buccal gingiva of the cat resulted in retrograde labeling in the ipsilateral otic ganglion (OG), whereas labeled neurons appeared in the pterygopalatine ganglion (PPG) as well as in the OG when the tracer was injected into the maxillary lip and buccal gingiva. The results suggest that both the facial and the glossopharyngeal preganglionic vasodilator fibers supplying the mandibular lip and buccal gingiva mediated in the OG, and those innervating the maxillary lip and buccal gingiva are mediated in the PPG and the OG.

Intraocular projections from the pterygopalatine ganglion in the cat.

The intraocular projection of the cat pterygopalatine (sphenopalatine) ganglion was examined by using retrograde axoplasmic transport techniques in order to investigate the possibility of the involvement of the facial nerve in ocular parasympathetic innervation. Following an injection of horseradish peroxidase (HRP) or wheat germ agglutinin-HRP into the eye, retrogradely labeled cells were observed in the ipsilateral pterygopalatine ganglion, principally in the caudal part. By dissection of silver-impregnated, acetylcholinesterase- and cholinesterase-stained orbital preparations, it was determined that two different nerve pathways link the pterygopalatine ganglion and the eye. One took a retrograde course to join the retro-orbital plexus and then traveled forward accompanying the ciliary artery, the long ciliary nerve, the short ciliary nerve, and/or the optic nerve sheath. The other entered the orbit directly, fused with the ethmoidal nerve or the infratrochlear nerve in a retrograde fashion, and then turned forward along the long ciliary nerve to enter the eye. All these nerves arose from the caudal part of the ganglion. These results are discussed in relation to recent biochemical and histochemical data demonstrating the involvement of the facial nerve in the control of ocular blood flow and intraocular pressure.

Morphology of the accessory ciliary ganglion of the cat.

A microdissection of the orbital nerves of the cat was made paying particular attention to the accessory ciliary ganglion. The structures were studied with the light microscope by histological and histochemical techniques. The accessory ciliary ganglion consisted of cells similar to those in the main ganglion. The neurons of the accessory ciliary and those of the main ciliary ganglion emitted no specific fluorescence for catecholamines. After injection of horseradish peroxidase-wheat germ agglutinin conjugates into the chambers of the eye, most ganglion cells of the accessory and main ciliary ganglia were labeled. A few labeled ectopic cells were also found in proximity of the accessory ganglion in the lateral short ciliary nerve or in the communicating branch from the trigeminal nerve. The present results indicate that the accessory ciliary and ectopic ganglion cells are parasympathetic in nature and may innervate the intrinsic eye musculatures.