Demonstration of a unique population of neurons with NADPH-diaphorase histochemistry

doi:10.1016/0165-0270(83)90085-7

Journal of Neuroscience Methods

Volume 9, Issue 3, November 1983, Pages 229-234

https://doi.org/10.1016/0165-0270(83)90085-7 Get rights and content

Abstract

A simple enzyme histochemical technique is described that detects various distinct populations of neurons in the brain. These neurons contain an extremely high activity of an endogenous enzyme, NADPH-diaphorase, that can reduce the dye nitro blue tetrazolium to a bright blue reaction product. Some of the major groups of neurons detected by this technique occur scattered throughout the neocortex, in the striatum and in the laterodorsal tegmental nucleus. The soma, dendritic trees and fiber networks of the positive neurons are stained in their entirety. Thus this simple, reliable technique can be used to obtain a Golgi-like image of particular groups of neurons in various regions of the brain.

References (13)

N. Shimizu et al.
Histochemical studies of the brain with reference to glucose metabolism
Progr. Brain Res.
(1966)
T. Abe et al.
Histochemical study of glucose-6-phosphate dehydrogenase in the brain of normal adult rat
Med. J. Osaka Univ.
(1963)
B. Falck et al.
Fluorescence of catecholamines and related compounds with formaldehyde
J. Histochem. Cytochem.
(1962)
R.L. Friede
A Histochemical Atlas of Tissue Oxidation in the Brain Stem of the Cat
(1961)
M.J. Karnovsky et al.
A ‘direct-coloring’ thiocholine method for acetylcholinesterases
J. Histochem. Cytochem.
(1964)
G.B. Koelle
The histochemical localization of cholinesterase in the central nervous system of the cat
J. comp. Neurol.
(1954)

There are more references available in the full text version of this article.

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Histological and neuronal changes in the duodenum of hamsters infected with Leishmania (Leishmania) infantum
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Visceral leishmaniasis is a neglected tropical disease caused by parasites belonging to the Leishmania genus that infect macrophages in different tissues such as the spleen, liver, lymph nodes, bone marrow, and intestine. Therefore, this study aimed to investigate the integrity of the intestinal tract and the nitrergic (NADPH-dp) and metabolically active (NADH-dp) myenteric neurons of the duodenum of golden hamsters infected with L. (L.) infantum. Therefore, thirty golden hamsters were divided into six groups (n = 5); three of them were infected with 2 × 10⁷ promastigote forms of L. (L.) infantum by intraperitoneal route (Infected Group - IG) and three were inoculated with saline solution (control group - CG). After 30, 60 and 90 days post-infection (DPI) infected animals were euthanized and the liver, spleen and duodenum were collected to analyze tissue parasitism. The duodenum was processed using usual histological techniques to analyze the main changes that occurred during infection and histochemical techniques to phenotype myenteric neurons. Amastigote forms were observed in the spleen, liver, and duodenum during all experimental periods, and tissue parasitism in these organs increased significantly over time. At 30 DPI, reduction in muscle tunic, increase in the total intestinal wall and the number of goblet cells PAS+ was observed. At 60 DPI, an increase in intestinal crypts and intraepithelial lymphocytes was observed, and a reduction in intestinal villi was observed at 90 DPI, along with an increase in crypt size. Regarding neurons, an increase in the density of the NADPH-dp population was observed at 30 DPI, but at 60 and 90 DPI a significant reduction of this population was observed. In general, infection progression was observed to cause significant morphofunctional changes in the duodenum of infected hamsters.
Histochemical and immunohistochemical localization of nitrergic structures in the carotid body of spontaneously hypertensive rats
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The carotid body (CB) is a multipurpose metabolic sensor that acts to initiate cardiorespiratory reflex adjustments to maintain homeostasis of blood-borne chemicals. Emerging evidence suggests that nitric oxide increases the CB chemosensory activity and this enhanced peripheral chemoreflex sensitivity contributes to sympathoexcitation and consequent pathology. The aim of this study was to examine by means of NADPH-diaphorase histochemistry and nitric oxide synthase (NOS) immunohistochemistry the presence and distribution of nitrergic structures in the CB of spontaneously hypertensive rats (SHRs) and to compare their expression patterns to that of age-matched normotensive Wistar rats (NWRs). Histochemistry revealed that the chemosensory glomus cells were NADPH-d-negative but were encircled by fine positive varicosities, which were also dispersed in the stroma around the glomeruli. The NADPH-d-reactive fibers showed the same distributional pattern in the CB of SHRs, however their staining activity was weaker when compared with NWRs. Thin periglomerular, intraglomerular and perivascular varicose fibers, but not glomus or sustentacular cells in the hypertensive CB, constitutively expressed two isoforms of NOS, nNOS and eNOS. In addition, clusters of glomus cells and blood vessels in the CB of SHRs exhibited moderate immunoreactivity for the third known NOS isoenzyme, iNOS. The present study demonstrates that in the hypertensive CB nNOS and eNOS protein expression shows statistically significant down-regulation whereas iNOS expression is up-regulated in the glomic tissue compared to normotensive controls. Our results suggest that impaired NO synthesis could contribute to elevated blood pressure in rats via an increase in chemoexcitation and sympathetic nerve activity in the CB.
Effect of Dp71 deficiency on the oxytocin hypothalamic axis in osmoregulation function in mice
2019, Acta Histochemica
Citation Excerpt :
Fluorescence was observed with a Zeiss Axioskop 2 Plus ﬂuorescence microscope (Carl Zeiss AG; Oberkochen, Germany), and images were captured with a Zeiss AxioCamHRc digital camera and Axiovision version 4.1 software (Carl Zeiss AG). NADPH-diaphorase staining was performed as described previously (Scherer-Singler et al., 1983). Briefly, slide mounted sections were incubated with 1 mM NAPDH, 0.2 mM nitroblue tetrazolium, 0.1 M Tris–HCl (pH 7.2), and 0.2% Triton X-100 for 1 h at 37 °C.
Dp71 is the major form of dystrophins (Dp) in the supraoptic nucleus (SON) and in the neural lobe of hypophysis (NL/HP). Dp71-null mice exhibit a hypo-osmolar status attributed to an altered osmosensitivity of the SON and to a perturbed vasopressinergic axis. Because oxytocin (OT) is implicated in osmoregulation via natriuresis, this study explored the oxytocinergic axis in Dp71-null mice after salt-loading (SL).
Under normosmolar conditions, OT-mRNA expression was higher in the Dp71-null SON compared to wild-type (wt) and the OT peptide level has not changed. Dp-immunostaining was localized in astrocytes end-feet surrounding vessels in wt SON. This distribution changed in Dp71-null SON, Dp being detected in OT-soma of MCNs. nNOS and NADPH-diaphorase levels increased in the OT area of the Dp71-null SON compared to wt. In the NL/HP, OT level reduced in Dp71-null mice and Dp localization changed from pituicytes end-feet in wt SON to OT terminals in Dp71-null SON.
Salt-Loading resulted in an increase of OT-mRNA and peptide levels in wt SON but had no effect in Dp71-null SON. In the NL/HP, OT content was reduced after SL. For Dp71-null mice, OT level, already low in control, was not modified by SL. Dp level was not affected by SL in the SON nor in the NL/HP.
Our data confirmed the importance of Dp71 for the SON functionality in osmoregulation. The localization of Dp71 at the glial-vascular interface could be associated with SON osmosensitivity, leading to an adequate OT synthesis in the SON and release from the NL/HP upon plasmatic hyperosmolality.
Enteric nervous system analyses: New biomarkers for environmental quality assessment
2018, Marine Pollution Bulletin
The gastrointestinal tract (GIT) of fish is a target of contaminants since it can absorb these substances. We evaluated the morphophysiological alterations in the GIT of Sphoeroides testudineus collected in two estuaries presenting differences in their environmental quality (NIA and IA). The intestine was analyzed for histological and neuronal changes; liver and gills for biochemical markers; muscle tissues for neurotoxicity and peripheral blood for genotoxic damage. The results showed alterations in the GIT of the animals collected in the IA, such as muscle tunica and goblet cell density reduction, increased intraepithelial lymphocytes density and changes in neuronal density. Furthermore, changes were observed in MTs and LPO in the gills. Thus, we suggest that TGI is functioning as a barrier that responds to ingested contaminants, in order to reduce their absorption and translocation. Thus, alterations in morphophysiological and enteric neurons in S. testudineus can be used as biomarkers of environmental contamination.
Toxoplasma gondii infection causes structural changes in the jejunum of rats infected with different inoculum doses
2017, Life Sciences
To evaluate the mucosal tunic and submucosal plexus of the jejunum of rats infected with different inoculum doses of Toxoplasma gondii.
Rats were infected with different inoculum doses (50, 500, 1000 and 5000 oocysts) of the T. gondii for 30 days, while a control group (CG) received saline solution. Blood and feces were collected before euthanasia for analysis of blood and fecal leukocytes (LEs). Histological analysis of the mucosa, submucosa, villi, crypts and enterocytes were performed. Goblet cells, intraepithelial lymphocytes (IELs) and Paneth cells were quantified. Immunohistochemistry was used to assess enteroendocrine serotonergic (5HT-IR) cells, proliferative cells (PCNA⁺) and mast cells. Whole mounts were obtained to determine the total submucosal neurons by Giemsa staining and metabolically active neurons (NADH-d⁺), nitrergic neurons (NADPH-d⁺) and glial cells (S100).
An increase in blood LEs was observed 30 days post-infection (dpi). Fecal LEs were more abundant in the feces in all infected groups at 21 dpi when compared to the CG. The number of IELs, sulfomucin-producing goblet cells, Paneth cells, PCNA⁺ cells and mast cells increased, whereas the number of 5HT-IR cells decreased. The jejunal architecture was altered, with atrophy of the mucosa, submucosa, villi and crypts. The number of total submucosal neurons decreased, but the NADPH-d⁺ subpopulation increased.
The results show how chronic toxoplasmic infection affects the tissue and cellular composition of the rat jejunum. These structural changes tend to intensify with the inoculum dose, demonstrating the importance of the parasitic load on intestinal alterations.
Acute infection with an avirulent strain of Toxoplasma gondii causes decreasing and atrophy of nitrergic myenteric neurons of rats
2017, Acta Histochemica
In the enteric nervous system (ENS), nitrergic neurons produce and use nitric oxide (NO) as an inhibitory motor neurotransmitter in response to parasitic infections, including those caused by Toxoplasma gondii. However, damage to the host caused by NO has been reported by various authors, and the role of NO in protection or cytotoxicity continues to be extensively studied. In this study, nitrergic neurons were investigated in the myenteric plexus of the jejunum and the distal colon of rats infected with 500 oocysts of the M7741 strain of T. gondii. Ten rats were randomly assigned into a control group (CG) and infected group (IG; received 500 sporulated oocysts of T. gondii orally). After 24 h, the rats were euthanized, and samples of the jejunum and distal colon were obtained and processed for NADPH-diaphorase histochemical analysis. Quantitative and morphometric analysis of the nitrergic neurons in whole mounts containing the myenteric plexus was performed. There was a numeric reduction of nitrergic neurons per mm² in both jejunum and distal colon. The remaining nitrergic neurons suffered atrophy in the areas of the cell body and nucleus, which resulted in a decrease in cytoplasm. Thus, we conclude that an avirulent strain of T. gondii in a short time causes neuroplastic changes in the small and large intestine of rats.

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^∗∗: Present address: Department of Anatomy, Shiga University of Medical Sciences, Ohtsu, Shiga, Japan.

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Research paperDemonstration of a unique population of neurons with NADPH-diaphorase histochemistry

Abstract

Progr. Brain Res.

Histochemical study of glucose-6-phosphate dehydrogenase in the brain of normal adult rat

Med. J. Osaka Univ.

Fluorescence of catecholamines and related compounds with formaldehyde

J. Histochem. Cytochem.

A Histochemical Atlas of Tissue Oxidation in the Brain Stem of the Cat

A ‘direct-coloring’ thiocholine method for acetylcholinesterases

J. Histochem. Cytochem.

The histochemical localization of cholinesterase in the central nervous system of the cat

J. comp. Neurol.

Research paper
Demonstration of a unique population of neurons with NADPH-diaphorase histochemistry