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Nitric oxide (NO) can have opposite effects on peripheral sensory neuron sensitivity depending on the concentration and source of NO, and the experimental setting. The aim of this study was to determine the role of endogenous NO production in the regulation of mechanosensitive Ca(2+) influx of dorsal root ganglion (DRG) neurons. Adult mouse DRG neurons were grown in primary culture for 2-5 days, loaded with Fura-2, and tested for mechanically mediated changes in [Ca(2+)](i) by fluorescent ratio imaging. In the presence of the NOS inhibitors L-NAME, TRIM, or 7-NI, but not the inactive analogue D-NAME, peak [Ca(2+)](i) transients to mechanical stimulation were increased more than 2-fold. Neither La(3+) (25 microM), an inhibitor of voltage activated Ca(2+) channels, or tetrodotoxin (TTX, 1 microM), a selective inhibitor of voltage-gated Na(+) channels, had an effect on mechanically activated [Ca(2+)](i) transients under control conditions. However, in the presence of L-NAME, both La(3+) and TTX partially blocked the [Ca(2+)](i) response. Addition of Gd(3+), a blocker of mechanosensitive cation channels and L-type Ca(2+) channels, at a concentration (100 microM) that markedly inhibited the mechanical response under control conditions, only partially inhibited the response in the presence of L-NAME. The combination of either La(3+) or TTX with Gd(3+) caused near complete inhibition of mechanically stimulated [Ca(2+)](i) transients in the presence of L-NAME. We conclude that focal mechanical stimulation of DRG neurons causes Ca(2+) influx occurs primarily through mechanosensitive cation channels under control conditions. In the presence of NOS inhibitors, additional Ca(2+) influx occurs through voltage-sensitive Ca(2+) channels. These results suggest that endogenously produced NO in cultured DRG neurons decreases mechanosensitivity by inhibiting voltage-gated Na(+) and Ca(2+) channels.

The purpose of this study was to determine whether there is a specific organization of the primary sensory innervation on to identified vascular neurons in the inferior mesenteric ganglion (IMG) in guinea-pig. Retrograde tracers were placed intraluminally in inferior mesenteric artery (IMA) or inferior mesenteric vein (IMV) in vitro to identify ganglionic neurons as arterial, venous or unlabeled neurons. The distribution of primary sensory nerve fibers containing calcitonin gene-related peptide (CGRP), neuronal nitric oxide synthase (NOS) and substance P immunoreactivity (SP-IR) was compared before and after treatment with capsaicin. In control animals the density of immunoreactivity varied both with the transmitter and the type of neuron innervated. The density of immunoreactivity for all the three substances was reduced by capsaicin treatment. The degree of reduction of immunoreactivity in the fibers varied with the transmitter and the type of neuron. The density of CGRP and SP immunoreactive fibers was greatest around unlabeled neurons; 78% of the CGRP fibers were of primary sensory origin and all of the SP fibers were primary sensory. Around arterial neurons 44% of the CGRP fibers were of primary sensory origin and around venous 68% were primary sensory. NOS positive innervation around venous neurons was denser than around arterial neurons and all of it was completely (97%) eliminated by capsaicin, indicating that it was solely of primary sensory origin. We conclude that the primary sensory fibers innervating the IMG are differentially distributed to arterial and venous neurons and that the pattern of distribution is characteristic for each sensory neurotransmitter., (Copyright 1999 Elsevier Science B.V.)

The distribution of nitric oxide synthase (NOS) immunoreactive nerve fibers in the carotid body was compared between normoxic and chronically hypoxic rats (10% O2 and 3.0-4.0% CO2 for 3 months). NOS immunoreactive fibers appeared as thin processes with many varicosities. They were distributed predominantly around small arteries and arterioles, and around clusters of glomus cells. When expressed by the density of varicosities per unit area in the parenchyma, the density of NOS fibers associated with the vasculature and with the glomus cells in the chronically hypoxic carotid bodies was significantly decreased. Because nitric oxide (NO) is an inhibitory neuronal messenger in the normoxic carotid body, the present findings suggest that the sensory mechanisms in the hypoxic carotid body may be involved in 'disinhibition' resulting from reduced NO synthesis., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

We compared the pattern of nitric oxide synthase (NOS) immunoreactivity in retinas of rats during normal development and after unilateral transection of the optic tract at postnatal day 7. NOS was first detected in the second postnatal week in the inner nuclear and inner plexiform layers. There was no detectable difference in the overall pattern of immunoreactivity between normal retinas and retinas with severe loss of ganglion cells due to the lesion. We suggest that NOS may have a role in synaptic and vascular development in the inner retina, but is unlikely to play a major role in normal physiological retinal ganglion cell death or axotomy-induced cell death.

NADPH-diaphorase histochemistry has been shown to stain cells which contain nitric oxide synthase, an enzyme responsible for the biosynthesis of the freely diffusable gas nitric oxide. A number of studies have mapped the distribution of NADPH-diaphorase-reactive neurons in the hippocampal formation but they have failed to yield consistent data. The major point of controversy concerns the presence of NADPH-diaphorase-reactive pyramidal cells in the CA1 subfield of the rat hippocampal formation. The present results show that CA1 pyramidal neurons do contain nitric oxide synthase (NOS) which can be reliably demonstrated with the appropriate histochemical procedure. One of the critical determinants of CA1 pyramidal cell NADPH-diaphorase activity is shown to be incubation of brains in sucrose solution prior to histochemical processing. Subicular pyramidal cells were also found to contain NOS and to possess NADPH-diaphorase activity. These results explain a number of contradictory reports in the literature relating to the presence of NADPH-diaphorase activity in hippocampal principal cells. Additionally, densitometric analysis carried out on 20 microns thick sections, from brains incubated in sucrose solution, indicated that there were characteristic gradients. The intensity of NADPH-diaphorase activity in pyramidal cells located in the ventral subiculum was found to be greater than those in the dorsal subiculum. A similar, yet marginal, trend was apparent for pyramidal cells in CA1 and CA3, as well as nonpyramidal cells in CA1. At both dorsal and ventral levels, NADPH-diaphorase-positive subicular pyramidal cells and CA1 nonpyramidal cells also show a greater intensity than CA1 or CA3 reactive pyramidal neurons. This study also showed that tissue incubation in sucrose solution prior to immunocytochemistry, enhanced immunoreactivity of the endothelial isoform of NOS whilst having little effect on neuronal NOS reactivity.

Nitric oxide synthase activity was studied by means of NADPH-diaphorase activity and nitric oxide synthase immunoreactivity in the main and accessory olfactory bulbs of the frog Rana perezi and the newt Triturus marmoratus. In both species, NADPH-diaphorase staining was observed in all olfactory fibers. Vomeronasal fibers were NADPH-diaphorase-positive in Triturus but they were NADPH-diaphorase-negative in Rana. Nitric oxide synthase immunoreactivity was not observed in the primary afferents in any case. Granule cells were NADPH-diaphorase-positive and nitric oxide synthase-immunopositive in the main and accessory olfactory bulb of Rana, and in the main olfactory bulb of Triturus. The homogeneous NADPH-diaphorase staining of olfactory fibers is similar to what has been reported in teleosts, and it contrasts with the spatial segregation of NADPH-diaphorase-positive and -negative olfactory projections in rodents. These results confirm the interspecies variability of the NADPH-diaphorase/nitric oxide synthase distribution in the olfactory system of vertebrates.

The present in vivo study showed the expression of nitric oxide synthase-like immunoreactivity in epiplexus cells in the lateral ventricles induced by intracerebral injection of lipopolysaccharide into postnatal rats. Nitric oxide synthase-like immunoreactivity was vigorously expressed in epiplexus cells 1 and 3 days after the lipopolysaccharide injection, but by 7 days post-injection, it became undetectable. The expression of nitric oxide synthase-like immunoreactivity was also observed in some of the choroid epithelial cells. The nitric oxide synthase-like immunoreactivity in these cells appeared to be more intense in the ventricle ipsilateral to the LPS injection than that on the contralateral side. The immunostaining patterns of OX-42 and OX-6 for the detection of complement type 3 receptors and major histocompatibility complex class II antigens respectively paralleled that of anti-nitric oxide synthase, indicating that lipopolysaccharide-induced nitric oxide synthase-like immunoreactivity was primarily in epiplexus cells. Immunoelectron microscopy revealed that the nitric oxide synthase-like immunoprecipitate in epiplexus cells and choroid epithelial cells filled the entire cytoplasm and in some areas associated with the membranes of some of the organelles especially the mitochondria, suggesting that the enzyme is mainly cytosolic. It is speculated that nitric oxide synthase in these cells is involved in the synthesis of nitric oxide. The nitric oxide production, if any, through the enzymatic activity of nitric oxide synthase in epiplexus cells as well as the choroid epithelial cells may be involved in host defense against bacterial endotoxin in the ventricular system of postnatal rat brain.

On the basis of the comparing of the distribution of beta-endorphin-like immunoreactive neuronal fibres and nitric oxide synthase-like immunoreactive neurons in the dorsal raphe nucleus, the synapses between the two immunocytochemically identified neurons were studied with a modified DAB-silver-gold intensification double immunostaining technique at the electron microscopic level. Although both of them can be found in the mediodorsal and medioventral parts of the dorsal raphe nucleus, the synapses between them could only be found in the mediodorsal part. The majority of the beta-endorphin-like immunoreactive neuronal fibers contained many dense-cored vesicles. The synapses made by beta-endorphin-like immunoreactive neuronal axon terminals on nitric oxide synthase-like immunoreactive neurons were both symmetrical and asymmetrical with the former predominant, especially in the axo-dendritic ones. beta-Endorphin-like immunoreactive perikarya could only be found in the ventrobasal hypothalamus. These findings suggest the possibility that the beta-endorphin- producing neurons in the ventrobasal hypothalamus could influence nitric oxide synthase-containing neurons in the dorsal raphe nucleus by synaptic relations.