Search

The enzyme nitric oxide synthase (NOS) which is necessary for the production of nitric oxide from L-arginine exists in three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Our previous studies have demonstrated the roles of nNOS and eNOS within the rostral (RVLM) and caudal ventrolateral medulla (CVLM) in modulating cardiovascular responses during static skeletal muscle contraction via altering localized glutamate and GABA levels (Brain Res. 977 (2003) 80-89; Neuroscience Res. 52 (2005) 21-30). In this study, we investigated the role of iNOS within the RVLM and CVLM on cardiovascular responses and glutamatergic/GABAergic neurotransmission during the exercise pressor reflex. Bilateral microdialysis of a selective iNOS antagonist, aminoguanidine (AGN; 1.0 microM), for 60 min into the RVLM attenuated increases in mean arterial pressure (MAP), heart rate (HR), and extracellular glutamate levels during a static muscle contraction. Levels of GABA within the RVLM were increased. After 120 min of discontinuation of the drug, MAP and HR responses and glutamate/GABA concentrations recovered to baseline values during a subsequent muscle contraction. In contrast, bilateral application of AGN (1.0 microM) into CVLM potentiated cardiovascular responses and glutamate concentration while attenuating levels of GABA during a static muscle contraction. All values recovered after 120 min of discontinuation of the drug. These results demonstrate that iNOS within the ventrolateral medulla plays an important role in modulating cardiovascular responses and glutamatergic/GABAergic neurotransmission that regulates the exercise pressor reflex.

The distribution of nitric oxide synthases (NOS) in the lower brain stem was studied by NADPH-diaphorase staining with emphasis on the nucleus of the solitary tract and the ventrolateral medulla. The order of NOS density was hamster > rabbit = rat > mouse > guinea-pig > cat with little variation within species or between regions. This heterogeneity may partly explain qualitatively and quantitatively variable effects of NO on sympathetic activity in different species.

Peripheral nerve injury in a rat model (spinal nerve ligation) of neuropathic pain triggers sprouting of sympathetic fibers in the dorsal root ganglion (DRG). This sympathetic sprouting has been suggested as an important underlying mechanism for pain behaviors. To investigate the possibility of functional interaction between sprouted sympathetic fibers and sensory neurons, the present study examined the fine morphology and structural relationship between sympathetic fibers and the DRG neurons by electron microscopy. Sympathetic postganglionic fibers, as identified by electron microscopic immunostaining for tyrosine hydroxylase (TH), were all unmyelinated fibers and some of them ended as growth cones. In addition, many vesicle-containing axonal enlargements (we will refer these as synaptic varicosities) were found in the interstitial space around DRG neurons, and some were enclosed within the satellite cell capsule which surrounded the DRG soma. The presence of sympathetic synaptic varicosities near or in apposition with either the DRG somata or their processes provides a structural basis for possible interactions between sensory neurons and sympathetic fibers in the DRG of neuropathic rats.

We studied the distribution of NADPH-d-reactive sympathetic preganglionic neurons (SPNs) and tyrosine hydroxylase (TH) immunoreactive fibers at T1, T4, T8, and T10 of human thoracic cord. NADPH-d-reactive SPNs were present at all segments. TH-immunoreactive fibers were distributed within the NADPH-d neuropil and appeared to contact SPNs. These interactions may be important for normal and pathological control of arterial pressure.

With retrograde tracing using fluorogold injection into the superior cervical ganglion and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry, the present comparative study revealed that the retrogradely labelled neurons in n. intermediolateralis pars funicularis (ILf) and n. intermediolateralis pars principalis (ILp) of the autonomic region in the upper thoracic cord exhibited a much stronger reactivity for NADPH-diaphorase in Wistar-Kyoto (WKY) rats than those in spontaneously hypertensive rats (SHR). It was found that in ILf in WKY rats, 77.62% of the fluorogold-labelled neurons were NADPH-d positive, while in SHR, only 56.43% of the labelled neurons were NADPH-d positive. The frequency distribution of NADPH-d positive retrogradely labelled neurons was significantly reduced in ILf of the spinal cord of SHR (U-test: P < 0.01). In ILp in WKY rats, 65.25% of fluorogold-labelled neurons were NADPH-d positive in WKY rats, while in SHR, only 56.28% of the labelled neurons were NADPH-d positive. Although the difference (P > 0.05) in the frequency of NADPH-d positive neurons in ILp between the two strains of rats was not significant, the reductions in SHR seemed considerable. Examination of the preganglionic sympathetic trunk and the superior cervical ganglion between SHR and WKY rats revealed that virtually all the NADPH-d positive fibers were derived from the sympathetic preganglionic neurons. In SHR, the NADPH-d positive fibers were not as intensely stained as those of WKY rats. This preliminary results suggest that nitric oxide, as an inhibitory neurotransmitter, may be implicated in the onset of hypertension.

Nitric oxide synthase (NOS)-immunoreactivity was co-localised with NADPH diaphorase activity in preganglionic sympathetic neurons and in their terminals in pre- and paravertebral sympathetic ganglia. The density of NOS-containing terminals varied between ganglia. Reactive terminals were densest in the superior cervical, stellate and inferior mesenteric ganglia, where the majority of the neurons were surrounded by reactive fibres, and the coeliac and superior mesenteric ganglia, where about half the postganglionic somata were surrounded by reactive terminals. Fibres were least abundant in the pelvic ganglia and thoracic and lumbar sympathetic chain ganglia. NOS reactivity did not coincide with the distribution of calcitonin gene related peptide immunoreactivity, a marker for the terminals of NOS-containing sensory neurons in the rat. The distribution of nerve cells and terminals suggests that NOS is present in more than one functional subpopulation of sympathetic preganglionic neurons.

The distribution of serotonergic nerves in major basal and isolated small pial arteries (diameter > or = 50 microns) was investigated immunohistochemically using an antibody directed against tryptophan-5-hydroxylase (TPOH), the rate-limiting enzyme in the synthesis of 5-hydroxytryptamine (5-HT or serotonin), and compared to that of the noradrenergic system labeled for the selective noradrenaline (NA) synthesizing enzyme, dopamine-beta-hydroxylase (DBH). In addition, the possible peripheral and/or central origins of the cerebrovascular serotonergic (TPOH-positive) nerve fibers were examined. Strongly labeled TPOH-immunoreactive (TPOH-I) fiber bundles were observed in major basal arteries and gave rise to small varicose fibers organized in a meshwork pattern. The highest density of TPOH-I fibers was found in the middle cerebral artery followed by the anterior cerebral and the anterior communicating arteries, with a moderate to low density in the internal carotid and the vertebro-basilar trunk. Of the isolated pial arteries, only the larger ones (diameter > 75 microns) were significantly endowed with TPOH-I varicose fibers. However, free floating TPOH-I nerves were observed coursing through the pia-arachnoid membranes and reaching small pial vessels. In contrast, DBH-I nerve fibers were fine and were visualized primarily as numerous varicosities distributed in a circumferential manner around the vessel wall. A very high density of DBH-I varicosities was seen in the rostral part of the circle of Willis, with the internal carotid being the most richly supplied followed by the anterior cerebral and the anterior communicating arteries; comparatively, the middle cerebral artery was moderately innervated. The differences in distribution pattern and density between TPOH-I and DBH-I cerebrovascular fibers clearly suggest that these two innervation systems are not exactly superimposable. Superior cervical ganglionectomy caused an almost complete disappearance of TPOH-I nerves in all vascular segments, with some residual fibers in selected vessels. Lesion of the central serotonergic component with the neurotoxin 5,7-dihydroxytryptamine had virtually no effect on the TPOH-I fibers in the major basal and isolated pial arteries. These results strongly suggest that the serotonergic innervation of major cerebral as well as pial arteries has a prominent peripheral origin closely related to the sympathetic system. Processing of superior cervical ganglion slices for TPOH immunocytochemistry, however, failed to unequivocally detect TPOH-I neurons.

This study examines possible changes in energy demands by developing neural crest cells in vitro using cytochrome oxidase (C.O.) histochemistry and immunohistochemical labeling of adrenergic (autonomic) cells and primary sensory neurons. Cytochrome oxidase is a key enzyme for oxidative metabolism and energy production, and it is used as a sensitive metabolic marker for neurons in the brain and dorsal root ganglia. In primary neural crest cell cultures, C.O. staining intensities differ among 4 distinct cellular populations (sensory neurons, adrenergic cells, pigment cells, and non-neuronal neural crest cells). At all stages, pigment cells exhibit extremely low C.O. staining. Neurons (both primary sensory and adrenergic cells) have higher C.O. activity than other cell types such as non-neuronal neural crest cells. This indicates that neurons have higher energy demands and presumably higher levels of functional activity than other cell types at least under the present culture conditions. All neurons in neural crest cell cultures elevate their energy demands during development, implying that energy metabolism and functional activity increase with neuronal maturation. At all stages, early determined sensory neurons exhibit more intense C.O. staining than late-developing sensory neurons. The difference in C.O. activity between the two populations of sensory neurons may be caused by different levels of functional activity due to their different time course of development. Tetrodotoxin (TTX) at 0.5-1 microM concentrations causes a decrease in the level of C.O. activity in the early determined sensory neurons, which may be correlated with a decrease in the functional activity of these neurons. Triple staining combining C.O. histochemistry with indirect immunofluorescence of antibodies against the stage specific embryonic antigen-1 (SSEA-1, which labels quail sensory neurons) and dopamine-beta-hydroxylase (DBH, which labels adrenergic cells) distinguish the level of C.O. activity between sensory neurons and autonomic cells. DBH+ cells exhibit relatively low C.O. staining. However, the C.O. activity among SSEA-1+ neurons varies from high to low levels. In general, SSEA-1+ sensory neurons are much more C.O. reactive than DBH+ autonomic cells. This suggests that developing sensory neurons in culture may have higher spontaneous and/or synaptic activity than autonomic neurons.

Markedly dilated dystrophic post-ganglionic sympathetic axons have been identified by dopamine-beta-hydroxylase immunohistochemistry in the paravascular ileal mesenteric nerves of rats with chronic streptozotocin diabetes. Many axons contained multiple dilatations with interposed axonal segments of near normal dimensions. These axonal abnormalities were absent in control animals. The time course of the development of the axonopathy and its distribution in the alimentary tract correlate with quantitative ultrastructural findings previously reported in this system.

Previous studies suggested the existence of two populations of cells in the mammalian sympathetic nervous system which differ in their ability to express phenylethanolamine N-methyltransferase (PNMT), the enzyme which specifically subserves the biosynthesis of epinephrine: (1) sympathoblasts and their progeny, the noradrenergic sympathetic neurons (PNMT negative); and (2) phaeochromoblasts , the precursors of the adrenergic cells of the adrenal gland and extra-adrenal chromaffin tissue (PNMT-positive). We sought to determine whether similar differences between sympathoblasts and phaeochromoblasts exist in other classes of vertebrate embryos. Using immunohistochemical and biochemical techniques to assay PNMT in sympathetic organs, we have found that chick embryo paravertebral ganglia contain PNMT activity both in vivo and in vitro. In vitro PNMT immunostaining was detected in principal neurons as well as in small process bearing neurons similar to mammalian SIF cells. In vivo, cells containing PNMT were seen not only in the adrenal gland, but also in other sympathetic structures such as the extra-adrenal chromaffin tissue and, unexpectedly, also in some cells of the kidney. Tyrosine hydroxylase, the first enzyme of the catecholamine (CA) biosynthetic pathway, was found in the same cells which could be stained with PNMT antibodies as well as in the sympathetic ganglia. Thus, we conclude that in contrast to the rat, chick sympathoblasts share with phaeochromoblasts the property of expressing all the CA enzymes, including PNMT.

Using a new coupled peroxidation method modified by adding nickel ammonium sulfate, we demonstrated the localization of monoamine oxidase (MAO) in the rat peripheral nervous system. MAO was localized in the endothelial cells of endoneurial vessels, in Schwann cell cytoplasm encircling myelinated axons, and in some unmyelinated axons. The morphometric ratio of these MAO-containing axons to the total unmyelinated axons was 10-13%. These MAO-containing unmyelinated axons were assumed to coincide with postganglionic sympathetic noradrenergic ones passing in the sciatic nerve. Histochemical MAO staining may be utilized to identify the postganglionic sympathetic nerves in normal and pathological conditions at the electron microscopic level in particular.

Glucocorticoids stimulate tyrosine hydroxylase activity and catecholamine synthesis, while markedly inhibiting acetylcholine synthesis and storage in the a clone of sympathetic nerve-like cells. Nerve growth factor enhances the effect of glucocorticoids on tyrosine hydroxylase. The steroid effect is specific for glucocorticoids, since 11-desoxycortisol, testosterone, and estradiol-17 beta do not reproduce the effects of hydrocortisone and dexamethasone. Concomitant with the shift in neurotransmitter synthesis, there is an increase in the mean diameter of intracellular dense core vesicles. In contrast to glucocorticoids, insulin increases the specific activity of choline acetyltransferase through the interaction with typical insulin receptors. Insulin does not, however, alter the morphology of the cells, nor does it block the morphological response of the cells to nerve growth factor.