Anatomic distribution of nitric oxide synthase in the heart

Nitric oxide synthase is a useful maker for nitric oxide's scope. Localized by either NADPH-diaphorase histochemistry or immunohistochemical methods, most nitric oxide synthase activity in the normal heart is present in endothelium along the extensive network of arteries, veins and capillaries within myocardium. This endothelial isoform of nitric oxide synthase also exists in the endocardium lining the cavities. Neuronal nitric oxide synthase appears much less prominent, although the exact amount of this isoform in the heart is uncertain. Scattered nerves and ganglion cells are localized by the histochemical methods. While there is no inducible nitric oxide synthase in the normal heart, macrophages associated with repair following various forms of cardiac damage contain this isoform. For all nitric oxide synthases, however, species variation and variability among models underscore the importance of correlative studies of structure and function.     

Coexistence of S100β and putative transmitter agents in vagal and glossopharyngeal sensory neurons of the rat

The coexistence of S100β with calcitonin gene-related peptide (CGRP), substance P (SP), somatostatin (SOM), nicotinamide adenosine dinucleotide phosphate-diaphorase (NADPH-d), and tyrosine hydroxylase (TH) was examined in the glossopharyngeal and vagal sensory ganglia. S100β immunoreactive (-ir) neurons in the jugular and petrosal ganglia frequently colocalized CGRP- or SP-ir, whereas S100β-ir neurons in the nodose ganglion infrequently contained CGRP- or SP-ir. No S100β-ir neurons in the jugular and petrosal ganglia showed SOM-ir while the small number of SOM-ir neurons in the nodose ganglion colocalized S100β-ir. Many neurons in the nodose ganglion colocalized S100β-ir and NADPH-d activity, whereas S100β-ir neurons in the jugular and nodose ganglia infrequently contained NADPH-d activity. S100β- and TH-ir were frequently colocalized in nodose ganglion but not in petrosal or jugular ganglion neurons. These findings suggest relationships between S100β and specific putative transmitters in functions of subpopulations of vagal and glossopharyngeal sensory neurons.     

Patterns of neuronal degeneration in the motor cortex of amyotrophic lateral sclerosis patients

Summary We examined patterns of neuronal degeneration in the motor cortex of amyotrophic lateral selerosis (ALS) patients using traditional cell stains and several histochemical markers including neurofilament, parvalbumin, NADPH-diaphorase, ubiquitin, Alz-50 and tau. Three grades of ALS (mild, moderate, severe) were defined based on the extent of Betz cell depletion. Non-phosphorylated neurofilament immunoreactive cortical pyramidal neurons and non-pyramidal parvalbumin local circuit neurons were significantly depleted in all grades of ALS. In contrast, NADPH-diaphorase neurons and Alz-50-positive neurons were quantitatively preserved despite reduced NADPH-diaphorase cellular staining and dendritic pruning. The density of ubiquitin-positive structures in the middle and deep layers of the motor cortex was increased in all cases. Axonal tau immunoreactivity was not altered. These histochemical results suggest that cortical degeneration in ALS is distinctive from other neurodegenerative diseases affecting cerebral cortex. Unlike Huntington's disease, both pyramidal and local cortical neurons are affected in ALS; unlike Alzheimer's disease, alteration of the neuronal cytoskeleton is not prominent. The unique pattern of neuronal degeneration found in ALS motor cortex is consistent with non-N-methyl-Dxxx-aspartate glutamate receptor-mediated cytotoxicity.Supported in part by a Muscular Dystrophy Association Research Development grant     

Coexistence of calbindin D-28k and NADPH-diaphorase in vagal and glossopharyngeal sensory neurons of the rat

The presence and coexistence of calbindin D-28k-immunoreactivity (ir) and nicotinamide adenosine dinucleotide phosphate (NADPH)-diaphorase activity (a marker of neurons that are presumed to convert L-arginine to L-citrulline and nitric oxide) were examined in the glossopharyngeal and vagal sensory ganglia (jugular, petrosal and nodose ganglia) of the rat. Calbindin D-28k-ir nerve cells were found in moderate and large numbers in the petrosal and nodose ganglia, respectively. Some calbindin D-28k-ir nerve cells were also observed in the jugular ganglion. NADPH-diaphorase positive nerve cells were localized to the jugular and nodose ganglia and were rare in the petrosal ganglion. A considerable portion (33–51%) of the NADPH-diaphorase positive neurons in these ganglia colocalized calbindin D-28k-ir. The presence and colocalization of calbindin D-28k-ir and NADPH-diaphorase activity in neurotransmitter-identified subpopulations of visceral sensory neurons were also studied. In all three ganglia, calcitonin gene-related peptide (CGRP)-ir was present in many NADPH-diaphorase positive neurons, a subset of which also contained calbindin D-28k-ir. In the nodose ganglion, many (42%) of tyrosine hydroxylase (TH)-ir neurons also contained NADPH diaphorase activity but did not contain calbindin D-28k-ir. These data are consistent with a potential co-operative role for calbindin D-28k and NADPH-diaphorase in the functions of a subpopulation of vagal and glossopharyngeal sensory neurons.     

NADPH-diaphorase activity in the hypothalamic magnocellular neurosecretory nuclei of the rat

A histochemical study of the distribution of NADPH diaphorase activity in the hypothalamus of normal rats was carried out. Our study demonstrates the presence of NADPH-diaphorase activity in the circularis and anterior and posterior fornicals nuclei for the first time. Additionally, we confirm the presence of NADPH-diaphorase-stained neurons in the paraventricular (both magno- and parvicellular neurons) and supraoptic nuclei, as well as a population of isolated positive neurons (not included in any hypothalamic nuclei) located among the different nuclei. Because NADPH diaphorase has recently been shown to be a nitric oxide synthase, our study reveals a wide presence of this enzymatic activity in the hypothalamus of the rat.     

NADPH-diaphorase: a selective histochemical marker for striatal neurons containing both somatostatin- and avian pancreatic polypeptide (APP)-like immunoreactivities

Certain neurons in the brain are specifically and intensely stained by a histochemical method which demonstrates nicotinamide adenine dinucleotide phosphate NADPH-diaphorase activity. The cell types containing this enzyme in certain areas of the rat forebrain were examined by combining NADPH-diaphorase histochemistry with the indirect immunofluorescence technique. Neurons containing somatostatin- or avian pancreatic polypeptide (APP)-like immunoreactivities were found throughout the forebrain including the striatum and neocortex. These two neuropeptides were also found to coexist in many telencephalic neurons. After photography, the sections processed for immunohistochemistry were stained for NADPH-diaphorase activity by a histochemical method. It was found that within the striatum all of the neurons that were selectively stained by this technique also contained both somatostatin- and APP-like immunoreactivities. Also in the neocortex NADPH-diaphorase was found only in those neurons displaying somatostatin- or APP-like immunoreactivity. In other brain regions such as the nucleus laterodorsalis tegmenti, NADPH-diaphorase-containing cells did not contain these neuropeptides. The results indicate that NADPH-diaphorase histochemistry provides a simple, reliable, histochemical method to demonstrate those striatal neurons in which somatostatin- and APP-like immunoreactivities coexist. The selective occurrence of this enzyme within these neurons may provide a useful target for pharmacological studies of these neuropeptide-containing cells.     

Neuronal and inducible nitric oxide synthase immunoreactivity following serotonin depletion

Serotonin (5HT) modulates the development and plasticity of its innervation areas in the central nervous system (CNS). Astrocytic 5HT(1A) receptors are involved in the plastic phenomena by releasing the astroglial-derived neurotrophic factor S-100beta. Several facts have demonstrated that nitric oxide (NO) and the nitric oxide synthase enzyme (NOS) may also be involved in this neuroglial interaction: (i) NO, S-100beta and 5HT are involved in CNS plasticity; (ii) micromolar S-100beta concentration stimulates inducible-NOS (iNOS) expression; (iii) neuronal NOS (nNOS) immunoreactive neurons are functionally and morphologically related to the serotoninergic neurons; (iv) monoamines level, including 5HT, can be modulated by NO release. We have already shown that 5HT depletion increases astroglial S-100beta immunoreactivity, induces neuronal cytoskeletal alterations and produces an astroglial reaction, while once 5HT level is recovered, a sprouting phenomenon occurs [Brain Res. 883 (2000) 1-14]. To further characterize the relationship among nNOS, iNOS and 5HT we have analyzed nNOS and iNOS expression in the CNS after 5HT depletion induced by parachlorophenylalanine (PCPA) treatment. Studies were performed immediately after ending the PCPA treatment and during a recovery period of 35 days. Areas densely innervated by 5HT fibers were studied by means of nNOS and iNOS immunoreactivity as well as NADPH diaphorase (NADPHd) staining. All parameters were quantified by computer-assisted image analysis. Increased nNOS immunoreactivity in striatum and hippocampus as well as increased NADPHd reactivity in the striatum, hippocampus and parietal cortex were found after PCPA treatment. The iNOS immunoreactivity in the corpus callosum increased 14 and 35 days after the end of PCPA treatment. These findings showed that nNOS immunoreactivity and NADPHd activity increased immediately after 5HT depletion evidencing a close functional interaction between nitrergic and serotoninergic systems. However, iNOS immunoreactivity increased when 5HT levels were normalized, which could indicate one of the biological responses to S-100beta release.     

Expression of NADPH-diaphorase and colocalization with Fos in the brain neurons of the rat following visceral noxious stimulation

We used double staining immunocytochemical techniques to determine whether nitric oxide (NO) and Fos immunoreactivity induced by noxious visceral stimulation were colocalized in the neurons of the supraspinal areas. We observed a considerable increase in Fos-positive neurons in many brain areas after noxious stimulation but only 15% of the Fos-positive neurons colocalized Nicotinamide Adenine Dinucleotide Phosphate Diaphorase (NADPH-d). The NADPH-d positive cells showed perikarya and cytoplasmic processes laying next to or more frequently apposed to Fos-positive neurons. This anatomical finding supported the hypothesis that also at supraspinal level NO is released near the neurons specifically activated and diffuses through the source cells to act on adjacent neurons playing a role in the central processing of pain transmission and modulation.     

NADPH-Diaphorase in the central nervous system of the larval lamprey (Lampetra planeri)

The anatomy of the hippocampus, including the organization of its intrinsic neural circuits and afferents, is organized along a rostrocaudal axis. Dopamine D2 receptors are expressed in specific regions of the hippocampal complex (hippocampal subfields, entorhinal cortex, perirhinal cortex) and show differential expression along this axis. The dentate gyrus and CA3/CA4 subfields show higher numbers of D2 receptors in the rostral than in the caudal levels. In contrast, the subiculum shows the reverse gradient. We report here that Alzheimer's disease (AD) is associated with reduced expression of the dopamine D2 receptor, but the effects differ with respect to the rostrocaudal axis and area within the hippocampal complex. The number of D2 receptors is significantly reduced in the molecular layer of the dentate gyrus, CA3 subfield, and subiculum. For the dentate gyrus and subiculum, there were greater losses at more rostral levels. The CA3/CA4 subfields showed the greatest losses caudally. The entorhinal cortex, which shows only modest expression of D2 receptors in controls, does not exhibit reduced numbers in AD. The external laminae of the rostral perirhinal cortex showed more significant losses than more caudally in this cortical field. The regions showing loss of D2 receptors do not typically contain neuritic plaques, neurofibrillary tangles, or significant neuron loss. Thus other mechanisms must account for the unique gradient of D2 receptor loss in the hippocampus. The regions of reduced expression of dopamine D2 receptors do correlate well with the terminal zone of the dentate association pathway, the afferents from the anlygdala and perirhinal cortex, and the sources of those afferents within the amygdala and perirhinal cortex. The specific patterns of reduced D2 receptor expression in AD are likely to contribute significantly to the disrupted information flow into and out of the hippocampus and, thus, of functions subserved by this system. © 1994 Wiley-Liss, Inc.     

Ultrastructural localization of NADPH-diaphorase activity in the submucous ganglia of the guinea-pig intestine after vagotomy

The reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity in the axon terminals presynaptic to the submucous neurons of guinea-pig intestine following unilateral cervical vagotomy was studied by electron microscopy. The reaction product of diaphorase was localized only in the axon terminals that contained predominantly small agranular vesicles, and it was usually deposited around the vesicles. The terminals that contained predominantly large granular or flattened vesicles did not display any signs of diaphorase reactivity. Although there were only few diaphorase-positive submucous neurons in the small intestine, a considerable number of diaphorase-positive axon terminals was observed in the submucous ganglia of the small intestine in the control animals. Ten days after vagotomy, the quantitative study showed that when compared with the control animals, the number of diaphorase-positive terminals in the submucous ganglia of duodenum, mid-small intestine and colon in the vagotomized animals was reduced (P<0.05). When the NADPHd-positive terminals were examined in closer detail, it was found that only a small proportion of them showed signs of degeneration as evidenced by the swelling and vacuolation of their contents of mitochondria, with disrupted cristae and clumping of synaptic vesicles. It was therefore concluded that at least some of the diaphorasepositive axon terminals in the submucous ganglia of guinea-pig intestine originated from the vagus nerve.