Methylmercury intoxication and histochemical demonstration of NADPH-diaphorase activity in the striate cortex of adult cats

The effects of methylmercury (MeHg) on histochemical demonstration of the NADPH-diaphorase (NADPH-d) activity in the striate cortex were studied in 4 adult cats. Two animals were used as control. The contaminated animals received 50 ml milk containing 0.42 µg MeHg and 100 g fish containing 0.03 µg MeHg daily for 2 months. The level of MeHg in area 17 of intoxicated animals was 3.2 µg/g wet weight brain tissue. Two cats were perfused 24 h after the last dose (group 1) and the other animals were perfused 6 months later (group 2). After microtomy, sections were processed for NADPHd histochemistry procedures using the malic enzyme method. Dendritic branch counts were performed from camera lucida drawings for control and intoxicated animals (N = 80). Average, standard deviation and Student t-test were calculated for each data group. The concentrations of mercury (Hg) in milk, fish and brain tissue were measured by acid digestion of samples, followed by reduction of total Hg in the digested sample to metallic Hg using stannous chloride followed by atomic fluorescence analysis. Only group 2 revealed a reduction of the neuropil enzyme activity and morphometric analysis showed a reduction in dendritic field area and in the number of distal dendrite branches of the NADPHd neurons in the white matter (P<0.05). These results suggest that NADPHd neurons in the white matter are more vulnerable to the long-term effects of MeHg than NADPHd neurons in the gray matter.

NADPH-diaphorase activity in area 17 on the squirrel monkey visual cortex: neuropil pattern, cell morphology and laminar distribution

We studied the distribution of NADPH-diaphorase activity in the visual cortex of normal adult New World monkeys (Saimiri sciureus) using the malic enzyme "indirect" method. NADPH-diaphorase neuropil activity had a heterogeneous distribution. In coronal sections, it had a clear laminar pattern that was coincident with Nissl-stained layers. In tangential sections, we observed blobs in supragranular layers of V1 and stripes throughout the entire V2. We quantified and compared the tangential distribution of NADPH-diaphorase and cytochrome oxidade blobs in adjacent sections of the supragramular layers of V1. Although their spatial distributions were rather similar, the two enzymes did not always overlap. The histochemical reaction also revealed two different types of stained cells: a slightly stained subpopulation and a subgroup of deeply stained neurons resembling a Golgi impregnation. These neurons were sparsely spined non-pyramidal cells. Their dendritic arbors were very well stained but their axons were not always evident. In the gray matter, heavily stained neurons showed different dendritic arbor morphologies. However, most of the strongly reactive cells lay in the subjacent white matter, where they presented a more homogenous morphology. Our results demonstrate that the pattern of NADPH-diaphorase activity is similar to that previously described in Old World monkeys.

Localization of NADPH diaphorase activity in the human visual cortex

The present report describes the activity of NADPH-diaphorase (NADPHd) in area 17 of autopsied normal human visual cortex. Four human brains from autopsy tissue (4-8 h postmortem) were fixed by immersion in 4 per cent paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.2-7.4, or in 10 per cent formalin for 24 h. NADPHd histochemistry was done using the malic enzyme indirect method. The neurpile pattern of enzyme activity presented a clear six layer appearance. Cell morphology and the laminar distribution of 73 NADPHd-positive neurons are descrived. All neurons found in area 17 of human cortex were sparsely spiny or smooth cells, located in all cortical layers exept layer 4c. Quantitative analysis of the branching pattern of the dendritic tree was carried out. A symmetrical pattern was observed with no particular dendritic bias except for a few white matter and layer 1 cells. Larger dendritic fields were found in white matter cells when compared to the other corical layers. Comparison of cell densities for gray and white matters showed that 85 per cent of the NADPHd-positive neurons were located in the white matter. NADPH was colocalized with nitric oxide synthase which produces nitric oxide, a short-life neuromediator implicated in synaptic plasticity, neuroprotection, and neurotoxicity. thus, the spatial distribution of the NADPHd cells is important for posterior functional studies of the neuromediators in the brain

Biochemical characterization of NADPH diaphorase in the chick embryo retina: stimulation by calcium ions and inhibition by arginine analogs

Nitric oxide is an important intercellular messenger in the central nervous system. NADPH-diaphorase, reported to be identical to nitric oxide synthase, is prsent in specific groups of cells in several neural tissues, including the retina. We determined NADPH-diaphorase activity in homogenates of the chick embryo retina. The enzyme activity was measured spectophotometrically at 585 nm after incubating retinal total homogenates (100-150 µg protein) with 1mMNADPH and 0.5 mM nitroblue tetrazolium in 50 mMTris buffer, pH8.1, at 37ºC. NADPH-diaphorse was detected in 14-day old retinas and 53-65 per cent of the enzyme activity was inhibited by 3 mM NG-nitro-L-arginine (NARG), the arginine analog. One mM L-N5-(1-iminoethyl)ornithine (NIO) was the most potent inhibitor (63 per cent inhibition) while 3 mM NG-nitro-L-arginine methyl ester (NAME) (33 per cent inhibition) and I mMNG-monomethyl-L-arginine acetate (NMMA) (14 per cent inhbition) were less effective. Enzyme activity was increased by 48 per cent by 2 mM calcium chloride, and effect reversed by 1 mMEGTA or EDTA. Basal enzyme levels were also partially inhibited by the chelators, indicating the presence of calcium-dependent and -independent isoforms of nitric oxide synthase in the retina. The results show that the NADPH-diaphorase assay is sample and sensitive and that the different isoforms of nitric oxide synthase expressed in chick retinal cells during development can be demonstrated

Evidence for two independent mechanisms of gaba release induced by veratridine and glutamate in monolayer cultures of chick embryo retinal cells

GABA is a major inhibitory neurotransmitter in the central nervous system, including the retina. In the present paper we present evidence for the existence of two independent mechanisms for GABA release in cultured retina cells. Eight-day-old chick embryo retinas were dissociated and plated in 35-mm plastic dishes and cultured for 3 or 7 days at 37 grade C. An increase of 3 to 5-fold in GABA release was observed in cultures of 3 or 7 days in vitro preloaded with 0.5 uCi[3H} GABA and stimulated with glutamate (100 uM) or veratridine (100 uM). Tetrodotoxin (1 uM) blocked the release induced by veratridine but not by glutamate. In contrast, the non-N-methyl-D-aspartate (NMDA)glutamate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 100 uM) was able to inhibit GABA release promoted by glutamate but not by veratridine. These results indicate that depolarization of retinal cells byopening of voltage-dependent sodium channels or activation of non-NMDA glutamate receptors can trigger intracellular events that lead to calcium-independent GABA release