David S. Bredt

Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase

Nitric oxide is a free radical that has been recently recognized as a neural messenger molecule. Nitric oxide synthase has now been purified and molecularly cloned from brain. Using specific antibodies and oligonucleotide probes, we have localized brain nitric oxide synthase to discrete neuronal populations in the rat and primate brain. Nitric oxide synthase is exclusively neuronal, and its localization is absolutely coincident with NADPH diaphorase staining in both rat and primate.

Targeted disruption of the neuronal nitric oxide synthase gene

By homologous recombination, we have generated mice that lack the neuronal nitric oxide synthase (NOS) gene. Neuronal NOS expression and NADPH-diaphorase (NDP) staining are absent in the mutant mice. Very low level residual catalytic activity suggests that other enzymes in the brain may generate nitric oxide. The neurons normally expressing NOS appear intact, and the mutant NOS mice are viable, fertile, and without evident histopathological abnormalities in the central nervous system. The most evident effect of disrupting the neuronal NOS gene is the development of grossly enlarged stomachs, with hypertrophy of the pyloric sphincter and the circular muscle layer. This phenotype resembles the human disorder infantile pyloric stenosis, in which gastric outlet obstruction is associated with the lack of NDP neurons in the pylorus.

Transient nitric oxide synthase neurons in embryonic cerebral cortical plate, sensory ganglia, and olfactory epithelium

Neuronal nitric oxide synthase (NOS), visualized immunohistochemically or with NADPH diaphorase histochemistry, is transiently expressed in discrete areas of the developing rat nervous system. In the brain transient NOS expression occurs in the cerebral cortical plate. At E15-E19, the majority of cells in the plate stain, with their processes extending through the corpus striatum to the thalamus. This staining decreases after birth and vanishes by the 15th postnatal day. Neurons in olfactory epithelium also express NOS from E15 till early postnatal life. In embryonic sensory ganglia virtually all neuronal cells are NOS positive, whereas by early adulthood only 1% express NOS. By contrast to these areas of transient NOS expression, in other neuronal sites NOS staining appears after cell bodies cease dividing and cells extend processes, and the staining persists in adult life. The transient expression of neuronal NOS may reflect a role in developmental processes such as programmed cell death.

Possible Origins and Distribution of Immunoreactive Nitric Oxide Synthase-Containing Nerve Fibers in Cerebral Arteries

The distribution of perivascular nerve fibers expressing nitric oxide synthase (NOS)-immunoreactivity was examined in Sprague–Dawley and Long–Evans rats using affinity-purified rabbit antisera raised against NOS from rat cerebellum. NOS immunoreactivity was expressed within the endothelium and adventitial nerve fibers in both rat strains. Labeled axons were abundant and dense in the proximal anterior and middle cerebral arteries, but were less numerous in the caudal circle of Willis and in small pial arteries. The sphenopalatine ganglia were the major source of positive fibers in these vessels. Sectioning postganglionic parasympathetic fibers from both sphenopalatine ganglia reduced the density of NOS-immunoreactive (IR) nerve fibers by >75% in the rostral circle of Willis. Moreover, NOS-IR was present in 70–80% of sphenopalatine ganglion cells. Twenty percent of these neurons also contained vasoactive intestinal polypeptide (VlP)-immunoreactivity. By contrast, the superior cervical ganglia did not contain NOS-IR cells. In the trigeminal ganglion, NO-IR neurons were found chiefly within the ophthalmic division; ∼10–15% of neurons were positively labeled. Colocalization with calcitonin gene-related peptide (CGRP) was not observed. Sectioning the major trigeminal branch innervating the circle of Willis decreased positive fibers by ≤25% in the ipsilateral vessels. In the nodose ganglion, 20–30% of neurons contained NOS-immunoreactivity, whereas less than 1% were in the C2 and C3 dorsal root ganglia. Three human circles of Willis obtained at autopsy showed sparse immunoreactive fibers, chiefly within vessels of the posterior circulation. Postmortem delay accounted for some of the reduced density. Our findings indicate that nerve fibers innervating cerebral arteries may serve as a nonendothelial source of the vasodilator nitric oxide (NO). The coexistence of NOS and VIP within sphenopalatine ganglion cells raises the possibility that two vasodilatory agents, one, a highly diffusable short-lived, low-molecular-weight molecule, and the other, a polar 28 amino acid-containing peptide, may serve as coneuromediators within the cerebral circulation.

Nitric oxide synthase: Irreversible inhibition by L-NG-Nitroarginine in brain in vitro and in vivo

Inhibition of nitric oxide (NO) synthase activity by L-NG-Nitroarginine (NO2Arg) in brain preparations is not reversed by dialysis and is enhanced by prolonged preincubation of NO2Arg with the enzyme. By contrast, the weaker inhibition by NO2Arg of macrophage NO synthase is fully reversible. NO2Arg inhibits NO synthase activity in the brain after i.p. administration of 5 or 50 mg/kg. This in vivo inhibition also appears to be irreversible. The potent in vivo inhibition of central NO synthase by NO2Arg may facilitate studies of the physiologic function of NO as a neuronal messenger.

The localization of nitric oxide synthase in the rat eye and related cranial ganglia

Nitric oxide synthase is the biosynthetic enzyme for the free radical neurotransmitter nitric oxide. Using an affinity-purified antiserum, nitric oxide synthase was found to be localized to peripheral ocular nerve fibers, related cranial ganglia, and the retina of the rat. In the eye, nitric oxide synthase-like immunoreactive peripheral nerve fibers were visualized mainly in the choroid and about limbal blood vessels. The anterior uvea was quite sparsely innervated, and the cornea was negative. Many principal neurons in the pterygopalatine ganglion were immunoreactive for nitric oxide synthase while very few cells stained in the superior cervical and trigeminal ganglia. Virtually all nitric oxide synthase-like immunoreactive pterygopalatine cells were also immunostained for vasoactive intestinal polypeptide; nitric oxide synthase also partially co-localized with neuropeptide Y in some of the neurons of this ganglion. Pterygopalatine ganglionectomy significantly reduced the number of peripheral nitric oxide synthase-like immunoreactive nerve fibers in the eye. A variety of immunoreactive retinal cells were seen. Most cells in the inner nuclear layer or ganglion cell layer corresponded morphologically to amacrine cells and displaced amacrine cells. Interplexiform cells and occasional faintly stained cells in the outer portion of the inner nuclear layer also were visualized. Nicotinamide adenine dinucleotide phosphate diaphorase histochemistry generally stained cells of similar distribution but did reveal somewhat more extensive localizations in peripheral ocular tissues, the ciliary ganglion, and the retina, compared with nitric oxide synthase immunohistochemistry. Nitric oxide synthase thus localizes to peripheral ocular nerve fibers, chiefly parasympathetic in nature and derived from the pterygopalatine ganglion, and to several cell types in the retina. Nitric oxide probably acts as a choroidal vasodilator of parasympathetic origin in the eye; the neuropeptide co-localizations in the pterygopalatine ganglion suggest complex neuromodulatory interactions. The retinal localizations imply potential neurotransmitter functions for nitric oxide in this tissue.

Nitric oxide mediates the formation of synaptic connections in developing and regenerating olfactory receptor neurons

Nitric oxide (NO) is a diffusible free radical that functions as a second messenger and neurotransmitter. NO synthase (NOS) is highly and transiently expressed in neurons of the developing olfactory epithelium during migration and establishment of primary synapses in the olfactory bulb. NOS is first expressed at E11 in cells of the presumptive nervous layer of the olfactory placode. NOS immunoreactivity persists in the descendants of these cells that differentiate into embryonic olfactory receptor neurons (ORNs). Olfactory NOS expression in the ORN and in its afferents rapidly declines after birth and is undetectable by P7. Following bulbectomy, NOS expression is rapidly induced in the regenerating ORN and is particularly enriched in their outgrowing axons. Immunoblot and Northern blot analyses similarly demonstrate an induction of NOS protein and mRNA expression, respectively, the highest levels of which coincide with peaks of ORN regeneration. These data argue against a role for NO in odorant-sensitive signal transduction, but suggest a prominent function for NO in activity-dependent establishment of connections in both developing and regenerating olfactory neurons.

Immunohistochemical Localization of Nitric Oxide Synthase in the Autonomic Innervation of the Human Penis

An improved understanding of the physiology of penile erection has resulted from recent evidence that implicates nitric oxide as the principal mediator of erectile function. Previously, the neuroanatomy of erection in man was established with descriptions of the autonomic innervation of the pelvic organs and external genitalia. The basis upon which novel physiological concepts of erection relate to earlier neuroanatomical principles remains to be determined. In the present study these relationships were explored with nitric oxide synthase immunohistochemistry and reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry of select pelvic tissue specimens obtained from 4 men (3 at radical prostatectomy and 1 at autopsy). Nitric oxide synthase, the enzyme that catalyzes nitric oxide production, was identified in discrete neuronal locations, including the pelvic plexus, cavernous nerves and their terminal endings within the corporeal erectile tissue, branches of the dorsal penile nerves and nerve plexuses in the adventitia of the deep cavernous arteries. This distribution of nitric oxide synthase-containing nerves suggests that nitric oxide neuronally modulates local vascular smooth musculature of the penis. On this basis, nitric oxide is identified as a neuronal mediator of penile erection in man.

Ultrastructural localization of nitric oxide synthese immunoreactivity in guinea-pig enteric neurons

Electron microscopic immunocytochemistry was used to localize immunoreactivity for nitric oxide synthase (NOS) in whole-mount preparations of myenteric plexus and circular muscle from guinea-pig ileum. NOS immunoreactivity was patchily distributed in myenteric neurons and was not specifically associated with any subcellular organelle or with the plasma membrane. This localization leaves unanswered the question of how nitric oxide is stored and released. NOS immunoreactive fibres in the circular muscle were found closer than 100 nm to muscle cells. NOS immunoreactive nerve fibres made synaptic contacts with NOS immunoreactive and non-immunoreactive enteric neurons. These results indicate that nitric oxide may regulate the activity of both myenteric neurons and smooth muscle.

Messenger molecules in the cerebellum

As data accumulate, the mammalian brain reveals its complex and subtle synaptic mechanisms. In the simplest system, a neurotransmitter binds to the receptor portion of a molecular complex incorporating an ion channel and thus alters the membrane potential, leading to excitatory or inhibitory effects. In more complex systems, receptors are coupled to second messenger systems to generate signals of longer duration and to modulate more diverse molecular mechanisms. The cerebellar cortex has a relatively simple wiring diagram with the primary neurotransmitter of most inhibitory and excitatory synapses well established. The second messenger signalling systems are more complex and those of the cerebellar output, the Purkinje cells, are the best characterized. More recently, molecules that might act as neuromodulators, carrying messages between neurons and between neurons and glial cells, have been identified, such as endothelin and nitric oxide. The classic neurotransmitters and novel neuromodulators, together with second messenger-activated trophic factors, can interact in complex ways; in this review Christopher Ross, David Bredt and Solomon Snyder discuss how studies of cerebellar circuitry and biochemistry are revealing such interrelations.