The distribution of the neuronal type of nitric oxide synthase in the goldfish brain and spinal cord was investigated via NADPH-diaphorase histochemistry and immunocytochemistry using an antiserum raised against the purified mammalian enzyme. Many structures, including magnocellular neurosecretory cells, motoneurons, mesencephalic trigeminal neurons, and radial glial fibers, were stained by the NADPH-diaphorase reaction but were not immunoreactive. This nonspecific NADPH-diaphorase activity was strongly reduced after preincubation of the sections. Therefore, when sections were first reacted for immunofluorescence and, thereafter, stained for NADPH-diaphorase, a corresponding staining pattern was obtained that allowed the reliable localization of neuronal nitric oxide synthase based on both complementary staining methods. In the telencephalon, positive neurons were concentrated in the ventral and posterior parts of the area ventralis. Many intensely stained neurons were present in various diencephalic nuclei, including the nucleus centralis posterior and the ventromedial nucleus of the thalamus, the nucleus tori lateralis, the nucleus recessus lateralis, the nucleus tuberis posterior, and the central nucleus of the inferior lobe. In the midbrain, neurons containing nitric oxide synthase were located in the periventricular zone of the optic tectum, the nucleus vermiformis, and the nucleus reticularis mesencephali. Specific staining in the cerebellum was concentrated in Golgi cells. In the hindbrain, nitroxergic neurons were numerous in all four sensory nuclei of the trigeminus, in the facial lobe, the superior olive, the inferior reticular formation, and the medial general visceral nucleus of the vagus. The dorsal horn of the spinal cord was enriched with positive neurons. A few strongly stained cells were also present in the ventral horn. In conclusion, neurons capable of synthesizing nitric oxide occur throughout the teleost central nervous system. The presence of nitric oxide synthase in projection areas of most afferent nerves suggests a widespread involvement of nitric oxide in sensory information processing. The distribution of nitric oxide synthase-containing neurons in certain areas, e.g., the tectum opticum and the spinal cord, indicates an evolutionarily conserved pattern. Similar to the case in other vertebrates, there appears to be no comprehensive overlap between the distribution of nitric oxide synthase and that of any other chemically characterized neuronal population described thus far. However, strongly positive cell groups in the mesencephalic reticular formation suggest the idea of an evolutionarily conserved mesopontine cholinergic system coexpressing nitric oxide synthase.

NADPH-diaphorase, an enzyme catalyzed reaction thought to reflect the activity of nitric oxide synthase in the mammalian nervous system, was mapped in the brain of the chicken. Intensely stained neurons and fibers were found in most parts of the telencephalon, in particular in the neostriatum, paleostriatum augmentatum, olfactory tubercle, lobus parolfactorius, hyperstriatum accessorium, and hyperstriatum ventrale. Medial to the nucleus taeniae, an accumulation of stained cells was observed that appeared to merge with a band of stained neurons located dorsal to the occipitomesencephalic tract. These are considered to belong to the nucleus interstitialis of the dorsal olfactory projection. Further caudally, neurons with different staining intensities were found in the lateral hypothalamic area, lateral mammillary nucleus, periventricular organ, ventral tegmental area, medial spiriform nucleus, optic tectum, isthmooptic nucleus, mesencephalic trigeminal nucleus, interpeduncular nucleus, and central gray of the mesencephalon. A particularly dense cluster of NADPH-diaphorase positive neurons was located in the locus coeruleus. It is proposed that these might represent cholinergic cells intermingled with catecholaminergic neurons, thus forming the avian counterpart of the tegmental cholinergic nuclei of mammals. Several NADPH-diaphorase reactive neurons were seen in the parabrachial nucleus and medial and dorsal vestibular nucleus, as well as scattered in the reticular formation. In the caudal medulla, intensely stained cells were grouped around the central canal. Therefore the pattern of expression of NADPH-diaphorase, and thus possibly of nitric oxide synthase, within the avian and mammalian brain might be largely conserved.

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The distribution pattern of nitric oxide synthase (NOS) was investigated in the brain of the turtle by NADPH-diaphorase histochemistry. The specificity of the histochemical staining was tested by immunocytochemical colocalization with an antiserum specific for NOS. In the forebrain, neurons staining intensely for nitric oxide synthase were localized in the olfactory tubercle, the basal ganglia complex, the basal amygdaloid nucleus, suprapeduncular nucleus, and the posterior hypothalamic area. Many positive fibers course in a tract connecting the basal amygdaloid nucleus with the hypothalamus, corresponding to the stria terminalis. Bundles of nitroxergic fibers were seen to course at the ventromedial edge of the optic tract and to cross in the supraoptic decussation, apparently consisting of tectothalamic and thalamotectal fibers. In the midbrain, strongly NOS-positive neurons were present in the substantia nigra, the nucleus profundus mesencephali, the periventricular grey of the optic tectum, the laminar nucleus of the torus semicircularis, and the nucleus of the lateral lemniscus. The area of the locus coeruleus harbored an accumulation of intensely stained neurons, which, as in mammals, might represent a cholinergic cell group of the reptilian brainstem. In the cerebellum, strong staining was confined to bundles of afferent fibers running in the lower molecular and in the Purkinje cell layer. These axons appeared to include ascending projections from the dorsal funicular nucleus or the spinal cord. NOS-positive cells in the caudal brainstem were found in the cerebellar nuclei, in the superior vestibular nucleus, in the reticular nuclei, ventrolateral to the nucleus of the solitary tract, in the perihypoglossal, and in the dorsal funicular nucleus. Taken together, these results suggest that nitric oxide acts as a messenger molecule in different areas of the reptilian brain and spinal cord. In certain areas, the pattern of expression of NOS appears to have evolved before radiation of present mammalian, avian, and reptilian species.