Search

The visual system is a potential target for methylmercury (MeHg) intoxication. Nevertheless, there are few studies about the cellular mechanisms of toxicity induced by MeHg in retinal cells. Various reports have indicated a critical role for nitric oxide synthase (NOS) activation in modulating MeHg neurotoxicity in cerebellar and cortical regions. The aim of the present study is to describe the effects of MeHg on cell viability and NOS activation in chick retinal cell cultures. For this purpose, primary cultures were prepared from 7-day-old chick embryos: retinas were aseptically dissected and dissociated and cells were grown at 37 degrees C for 7-8 days. Cultures were exposed to MeHg (10 microM, 100 microM, and 1 mM) for 2, 4, and 6 h. Cell viability was measured by MTT method and NOS activity by monitoring the conversion of L-[H3]-arginine to L-[H3]-citrulline. The incubation of cultured retina cells with 10 and 100 microM MeHg promoted an increase of NOS activity compared to control (P < 0.05). Maximum values (P < 0.05) were reached after 4 h of MeHg incubation: increases of 81.6 +/- 5.3 and 91.3 +/- 3.7%, respectively (data are reported as mean +/- SEM for 4 replicates). MeHg also promoted a concentration- and time-dependent decrease in cell viability, with the highest toxicity (a reduction of about 80% in cell viability) being observed at the concentration of 1 mM and after 4-6 h of incubation. The present study demonstrates for the first time the modulation of MeHg neurotoxicity in retinal cells by the nitrergic system.

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 microgram MeHg and 100 g fish containing 0.03 microgram MeHg daily for 2 months. The level of MeHg in area 17 of intoxicated animals was 3.2 micrograms/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.

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 oxidase blobs in adjacent sections of the supragranular 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.

NADPH-diaphorase (NADPH-d) activity was studied comparatively in area 17 of four mammalian species, two primates and two rodents. Three brain hemispheres each from adult capuchin-monkeys, owl-monkeys, agoutis and guinea pigs were fixed with aldehyde fixatives by perfusion and 200 microns sections were submitted to NADPH-d histochemistry, using the indirect malic enzyme method. In all species studied the neuropil pattern of enzymes activity presented a clear layered appearance. In primates, histochemical staining was most intense in layer IVc, while in rodents the highest intensity of the neuropil reaction was in supragranular layers (II and III). Comparison of cell density in grey and white matter showed that the majority of NADPH-d-positive neurones were located in the white matter of primates but not of rodents. Since NADPH-d is a nitric oxide synthase the results are very important for comparative functional studies of neuromediators and their correlations with laminar and modular organization of area 17 of the mammalian brain.

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% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.2-7.4, or in 10% formalin for 24 h. NADPHd histochemistry was done using the malic enzyme indirect method. The neuropile pattern of enzyme activity presented a clear six-layer appearance. Cell morphology and the laminar distribution of 73 NADPHd-positive neurons are described. All neurons found in area 17 of human cortex were sparsely spiny or smooth cells, located in all cortical layers except 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 cortical layers. Comparison of cell densities for gray and white matters showed that 85% of the NADPHd-positive neurons were located in the white matter. NADPHd 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.

Nitric oxide is an important intercellular messenger in the central nervous system. NADPH-diaphorase, reported to be identical to nitric oxide synthase, is present 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 spectrophotometrically at 585 nm after incubating retinal total homogenates (100-150 micrograms protein) with 1 mM NADPH and 0.5 mM nitroblue tetrazolium in 50 mM Tris buffer, pH 8.1, at 37 degrees C. NADPH-diaphorase was detected in 14-day old retinas and 53-65% 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% inhibition) while 3 mM NG-nitro-L-arginine methyl ester (NAME) (33% inhibition) and 1 mM NG-monomethyl-L-arginine acetate (NMMA) (14% inhibition) were less effective. Enzyme activity was increased by 48% by 2 mM calcium chloride, an effect reversed by 1 mM EGTA 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 simple and sensitive and that the different isoforms of nitric oxide synthase expressed in chick retinal cells during development can be demonstrated.

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 degrees 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 microCi [3H]GABA and stimulated with glutamate (100 microM) or veratridine (100 microM). Tetrodotoxin (1 microM) 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 microM) was able to inhibit GABA release promoted by glutamate but not by veratridine. These results indicate that depolarization of retinal cells by opening of voltage-dependent sodium channels or activation of non-NMDA glutamate receptors can trigger intracellular events that lead to calcium-independent GABA release.

1. The topographical distribution of ganglion cells and displaced amacrine cells in the guinea pig retina is described. 2. Neurons were counted in the ganglion cell layer of retinal whole mounts stained by the method of Nissl or retrogradely labeled with horseradish peroxidase. Neuronal soma size was estimated from samples taken from different retinal regions. 3. We estimate that a total of 295,000 neurons comprise the guinea pig ganglion cell layer and they consist of 159,000 ganglion cells and 136,000 displaced amacrine cells. 4. The visual streak is poorly differentiated. Ganglion cell density reaches a peak of 2,272 cells/mm2 in a temporal expansion of the visual streak, 4-5 mm toward the optic disk. The visual streak temporal expansion may represent the analogue of the area centralis for this species. The ventral hemi-retina has a higher ganglion cell density than the dorsal hemi-retina. The displaced amacrine cells are more uniformly distributed than the ganglion cells. 5. The present paper provides relevant data concerning the number and distribution of the neurons of the retinal ganglion cell which were not available or were very contradictory in the literature.