Nitric oxide regulates the proliferation of chick embryo retina cells by a cyclic GMP-independent mechanism

Nitric oxide (NO) is an intercellular messenger involved in many physiological and pathological processes of vertebrate and invertebrate animal tissues. In the embryonic chick retina, nitric oxide synthase (NOS) activity and a system for l-arginine transport between neurons and glial cells were described, supporting the idea that nitric oxide is a critical molecule during retinal development. In the present work we show that nitric oxide is a modulator of cell proliferation in chick embryo retina. Mixed cultures of retinal neurons and glial cells were submitted to [(3)H]-thymidine incorporation after drug treatment. Incubation for 24h with the NO donors S-nitroso-N-acetyl-penicillamine (SNAP) or Spermine nitric oxide (SpNO) complex promoted a decrease of approximately 70% of [(3)H]-thymidine incorporation in a dose-dependent manner. SNAP did not increase Lactate dehydrogenase release and its effect was not mimicked by 8-bromo cyclic GMP, or blocked by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), indicating that the effect was not due to cell death or mediated by increases of cyclic GMP levels. The inhibition was completely prevented by dithiotreitol (DTT), strongly indicating the participation of an S-nitrosylation mechanism. SNAP blocked the increase of [(3)H]-thymidine incorporation induced by ATP. Using purified cultures of glial cells we showed that the NO donor SNAP produced an inhibition of 50% in cell proliferation and did stimulate ERK1/2 phosphorylation, indicating that the inhibition of this pathway was not involved in its cytostatic effect. [(3)H]-Thymidine autoradiography of mixed cultures showed labeling of oval nuclei of glial flat cells. The injection of eggs with SNAP also did promote an intense inhibition of [(3)H]-thymidine incorporation in retinas from 9-day-old embryos. These data suggest that nitric oxide affects the proliferation of chick embryo retina glial cells in culture or "in vivo" through cyclic GMP and ERK-independent pathways.     

Developmental expression of nitric oxide synthase isoform I and III in chick retina

During our studies on the multiple possible functions of nitric oxide (NO) in chick retinal development and physiology, we have demonstrated the presence and the activity of NO synthase (NOS-I and III) in certain neuronal populations (photoreceptors, amacrine cells in the inner nuclear and ganglion cells) and also in synaptic-rich regions in the developing chick retina. Both enzymes, detected by nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, immunohistochemistry and Western blotting, appeared between embryonic days 6 and 12, and followed a spatial and temporal pattern of expression which correlated with the differentiation of the neuronal layers. Evaluation of the conversion of [³H]-labeled arginine to [³H]-citrulline, confirmed the presence of a calcium-dependent NOS activity in the cytosolic and particulate retinal extracts during the development. This pattern of NOS expression suggests that the regulated release of NO during key phases of development might be one mechanism involved in the regulation of retinal differentiation. J. Neurosci. Res. 50:104–113, 1997. © 1997 Wiley-Liss, Inc.     

Vasoactive intestinal peptide and nitric oxide promote survival of adult rat myenteric neurons in culture

Several motility disorders originate in the enteric nervous system (ENS). Our knowledge of factors governing survival of the ENS is poor. Changes in the expression of vasoactive intestinal peptide (VIP) and nitric oxide synthase (NOS) in enteric neurons occur after neuronal injury and in intestinal adaptation. The aim of this study was to evaluate whether VIP and nitric oxide (NO) influence survival of cultured, dissociated myenteric neurons. Neuronal survival was evaluated after 0, 4, and 8 days in culture. Influence of VIP and NO on neuronal survival was examined after culturing in the presence of VIP, NO donor, VIP antiserum, or NOS inhibitor. A marked loss of neurons was noted during culturing. VIP and NO significantly promoted neuronal survival. Corroborating this was the finding of an enhanced neuronal cell loss when cultures were grown in the presence of VIP antiserum or NOS inhibitor.     

Involvement of P2 purinoceptors in the regulation ofdna synthesis in the neural retina of chick embryo

The activation of P2 purinoceptors induces Ca2+ mobilization in the early embryonic chick neural retina. This purinergic Ca2+ response declines parallel with the decrease in mitotic activity during retinal development. To investigate the role of P2 purinoceptors in the regulation of retinal cell proliferation, we studied the effects of the P2 purinoceptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), and of the agonist ATP on DNA synthesis in retinal organ cultures from embryonic day 3 (E3) chick. Suramin inhibited [3H]-thymidine incorporation in a dose-dependent manner (IC50: approximately 70 microM). PPADS also reduced [3H]-thymidine incorporation with maximum inhibition of 46% at 100 microM. Exogenous ATP enhanced [3H]-thymidine incorporation in a dose-dependent manner to maximally 200% of control (EC50: approximately 70 microM). In dissociated retinal cultures from E7 chick, both antagonists showed similar inhibitory effects on [3H]-thymidine incorporation without affecting cell viability. In line with these observations, the presence of extracellular ATP was demonstrated both in vitro and in vivo. In the medium of E3 retinal organ cultures, the concentration of ATP increased 25-fold within 1 h of incubation and this concentration was kept for at least 24 h. In the chick amniotic fluid, the ATP concentration was nearly 3 microM at E3 and declined to 0.15 microM at E7. The results indicate that P2 purinoceptors activated by autocrine or paracrine release of ATP are involved in the regulation of DNA synthesis in the neural retina at early embryonic stages.     

Developmentally regulated release of intraretinal neurotrophic factors in vitro

The effects of conditioned media either from aggregates or from explants of embryonic chick retinae and of recombinant neurotrophins were tested upon the survival in vitro of ganglion cells in dissociated cell cultures from the retina of newborn rats. Ganglion cells were identified by the detection of retrogradely transported horseradish peroxidase injected bilaterally into the superior colliculus. Conditioned media increased significantly the survival of ganglion cells after 2 days in culture, at a wide range of plating densities, and had no effect upon adhesion of rat retinal cells. Media conditioned by cell ensembles from chick retinae from embryonic day 8 (E8) to E16 had neurotrophic effects. Release of neurotrophic activity peaked at E10-E12, irrespective of the numbers of cells or total concentration of protein in the conditioned media. The active molecules were non-dialyzable and were released either in the presence or in the absence of fetal calf serum. The neurotrophic activity was abolished by trypsinization, and recovered by salting-out with 25–75% ammonium sulfate. NT-4, BDNF and, to a lesser extent, NT-3, increased the survival of ganglion cells in our assay, while NGF had no effect. The data show that chick retinal cells release soluble trophic proteins according to a developmentally regulated pattern. These neurotrophic factors may be involved in local competitive interactions that help control naturally occurring neuron death among ganglion cells of the vertebrate retina.     

Nitric oxide is involved in sustained and delayed cell death of rat retina following transient ischemia

We have investigated the role of nitric oxide (NO) in the rat retina following ischemic injury induced by transient increase of intraocular pressure. The thickness of both the inner plexiform layer and inner nuclear layer decreased during early postischemic stages (up to 1 week). In late postischemic stages (2-4 weeks), the thickness of the outer nuclear layer (ONL) decreased markedly. Thus, mechanisms other than excitotoxic ones may contribute to postischemic retinal cell death. Treatment of rats with N(G)-nitro-L-arginine methyl ester, a nitric oxide synthase (NOS) inhibitor, significantly reduced ischemic damage. Our findings suggest that NO is involved in the mechanism of ischemic injury, and plays a key role in the delayed and sustained cell death in the ONL following transient retinal ischemia.     

Glutamate receptors coupled to nitric oxide synthesis in embryonic retina

Existing evidence suggests a role for nitric oxide (NO) in the establishment of stable synaptic connections during the embryonic development of the central nervous system. In the visual system, the participation of NO in programmed cell death, the natural elimination of photoreceptors and the modulation of photoreceptor ion channels has been documented. In the present work, the effect of ionotropic and metabotropic glutamate receptor (GluR) agonists on NO synthesis in the chick retina was studied at embryonic day (ED) 14, in which active synaptogenesis takes place, and compared with that in the mature tissue. We showed that at ED 14, N-methyl-D-aspartate, kainate and trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylate stimulate NO formation at low concentrations, whereas high concentrations of the drugs inhibit NO synthesis. The results suggest the participation of GluR-induced NO synthesis in differentiation and synaptogenesis, and demonstrate a negative feedback of GluR stimulation on NO synthesis by nonphysiological glutamate concentrations, which could be relevant to neuronal protection from glutamate-induced toxicity in the retina during a critical period of embryonic development.     

Ca2+ mobilization and capacitative Ca2+ entry regulate DNA synthesis in cultured chick retinal neuroepithelial cells.

Release of Ca2+ from intracellular Ca2+ stores (Ca2+ mobilization) and capacitative Ca2+ entry have been shown to be inducible in neuroepithelial cells of the early embryonic chick retina. Both types of Ca2+ responses decline parallel with retinal progenitor cell proliferation. To investigate their potential role in the regulation of neuroepithelial cell proliferation, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), an inhibitor of the Ca2+ pump of intracellular Ca2+ stores, and of SK&F 96365, an inhibitor of capacitative Ca2+ entry, on DNA synthesis in retinal organ cultures from embryonic day 3 (E3) chicks and in dissociated cultures from E7 and E9 chick retinae. We demonstrate that both antagonists inhibit [3H]-thymidine incorporation in a dose-dependent manner without affecting cell viability or morphology. The inhibition of [3H]-thymidine incorporation by SK&F 96365 occurred in the same concentration range (IC50: approximately 4 microM) as the blockade of capacitative Ca2+ entry in the E3 retinal organ culture. At a concentration of 5 microM SK&F 96365. DNA synthesis was reduced by 71, 40 and 32% in the E3, E7 and E9 cultures, respectively. Application of DBHQ at concentrations which led to depletion of intracellular Ca2+ stores also inhibited [3H]-thymidine incorporation with IC50 values of 20-30 microM in the different cultures. Our results suggest the involvement of Ca2+ mobilization and capacitative Ca2+ entry in the regulation of DNA synthesis in the developing neural retina.     

Initial cholinergic differentiation in embryonic chick retina is responsive to insulin and cell-cell interactions

Previous work [Kyriakis et al., Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 7463-7467] had shown that insulin, when added during a window of binding from embryonic days 9-11, stimulates the normal developmental increase in choline acetyltransferase (ChAT) activity (a marker for cholinergic differentiation) in cultured embryonic chick retinal neurons. Here, we investigated the effect of insulin and IGF 1 on embryonic chick retinal neurons at the stage of development (embryonic day 6) when ChAT activity is first expressed. We investigated insulin peptide effects in retinal tissue developing in vitro as well as in cultures of retinal cells. We show that insulin also stimulated the initial embryonic increase in ChAT activity but had no stimulatory effect on glutamic acid decarboxylase activity (a marker for GABAergic differentiation), an enzyme whose activity also increases developmentally in the same retinal neurons. In fact, insulin inhibited the expression of GAD activity in the retina. The insulin-mediated increase in ChAT activity was independent of normal cell-cell interactions but could not replace them. Insulin also stimulated choline uptake but only after a two day delay, suggesting that the normal program for cholinergic differentiation in the chick retina was induced by insulin. IGF 1 did not have any effect on either cholinergic or GABAergic differentiation. We conclude that cholinergic differentiation in chick embryo retinal neurons is dependent on both insulin- and cell contact-mediated signals.     

In situ localization of nitric oxide synthase and direct evidence of NO production in rat retinal ganglion cells

The expression of isoforms of nitric oxide synthase (NOS), enzymes responsible for NO production, and the synthesis of nitric oxide (NO) in rat retinal ganglion cells (RGCs) during synaptogenesis for various phases of the pre- and postnatal developmental periods were investigated. The retinas from prenatal, lactating, young, and adult rats were fixed in paraformaldehyde. The cryosections or paraformaldehyde-fixed ganglion cells purified from rat pups were immunostained for constitutive isoforms of NOS (n and eNOS) and observed with a confocal laser scanning microscope. Synthesis of NO in the RGCs was achieved by in vitro stimulation with glutamate. The intracellular NO levels were measured in real time using diaminofluorescein-2 diacetate, a fluorescence indicator of NO. Immunohistochemical analysis revealed nNOS and eNOS expressed in retinal ganglion cells during the first 2 postnatal weeks. Cultured RGCs also expressed nNOS and eNOS in vitro. Intracellular NO levels in cultured RGCs showed spontaneous fluctuation during a 20-min observation. The presence of both a non-specific NOS inhibitor, L-NAME, and a specific nNOS inhibitor, 7-NI, significantly inhibited (P<0.001) the increase of intracellular NO 6 and 8 min after the introduction of L-arginine and glutamate to the medium. This study revealed that all constitutive NOS isoforms are expressed in RGCs and demonstrated that NO is produced by nNOS mainly through stimulation by glutamate in cultured RGCs.     

Novel organization of microtubules in cultured central nervous system neurons: formation of hairpin loops at ends of maturing neurites

Using the high voltage electron microscope, we have examined cultured embryonic neurons in order to understand better the organization of microtubules in developing neurites. We found that, in embryonic chick retina neurons, microtubules were abundant in the ends of neurites and showed an unusual pattern of organization. Most striking was the presence of microtubule loops; after entering the flattened region of a growth cone, microtubules frequently made tight 180 degrees turns. Occasionally these looping microtubules re-entered the neurite and returned in the direction of the cell body. Positive identification of the loop structures as microtubules was made by specific immunocytochemical labeling. Quantitative analysis showed that more than half of the retina neurons that were dissociated on embryonic day 8 and kept in culture for 4 to 6 days (E8C4 and E8C6) contained at least one microtubule that made a 180 degrees turn at flat regions along or at the tips of neurites. The area within the loops typically contained larger membranous organelles, whereas only small vesicles were seen outside the loops. Fine filaments were seen to interconnect the loops at various places, suggesting the possibility that they played a role in maintaining the shape of microtubule loops. Examination of other neurons showed that tight microtubule loops were prominent in chick spinal cord neurons, but they were rarely seen in neurons of the sympathetic ganglia or dorsal root ganglia or in NG108-15 cloned cells. Developmentally, no loops were observed in E8C1 retina neurons, but retina neurons dissociated from older embryos (12 days) did show loops after 1 day in culture; these data suggest that microtubule loops may be abundant around embryonic day 12 to 13 in the chick retina. The possible significance of this unusual microtubule organization to the control of neurite growth and bidirectional transport is discussed.     

Nitric oxide synthase in developing retinas and after optic tract section

We compared the pattern of nitric oxide synthase (NOS) immunoreactivity in retinas of rats during normal development and after unilateral transection of the optic tract at postnatal day 7. NOS was first detected in the second postnatal week in the inner nuclear and inner plexiform layers. There was no detectable difference in the overall pattern of immunoreactivity between normal retinas and retinas with severe loss of ganglion cells due to the lesion. We suggest that NOS may have a role in synaptic and vascular development in the inner retina, but is unlikely to play a major role in normal physiological retinal ganglion cell death or axotomy-induced cell death.     

Increased expression of nitric oxide synthase in visual structures of the chick brain after retinal removal

Nitric oxide (NO) seems to act as a retrograde messenger in the establishment and refinement of synaptic connections during development and in neural plasticity processes in adult life. Previous studies have shown that the expression of NO synthase (NOS) in the optic tectum of developing chicks is regulated by the retinal innervation. The aim of this study was to observe the effects of unilateral retinal lesions upon the expression of NOS in central visual areas of the adult chick brain. After different survival times (1-30 days), the chick brains were submitted to immunohistochemical, immunoblotting, and NO imaging procedures to evaluate NOS expression and activity. Our results indicate that NOS expression in visual areas is also regulated by retinal innervation in the adult chick. However, differently from the case in the developing animal, the deafferentation seems to generate an increase of the NOS expression in retinorecipient visual areas. Our results suggest that NOS expression in visual structures of the adult chick brain may be down-regulated by the retinal innervation. Alternatively, the increase of NOS expression observed after retinal removal could be an indicative of a role of the NO system in plasticity processes. Copyright 2004 Wiley-Liss, Inc.     

Bax and caspases are inhibited by endogenous nitric oxide in dorsal root ganglion neurons in vitro

Axotomised dorsal root ganglia (DRG) neurons show an increased expression of neuronal nitric oxide synthase (nNOS) compared with neurons from the intact ganglia. Increased nNOS expression resulted in synthesis of nitric oxide (NO) and the subsequent activation of cGMP in satellite glia cells surrounding the DRG neuron soma. In dissociated DRG we have demonstrated that the increase in nNOS expression is regulated by nerve growth factor and that the subsequent inhibition of NO production or cGMP synthesis precipitates apoptosis of neurons expressing nNOS and some non-nNOS neurons. Hence, NO or the NO-cGMP cascade appears to have a neuroprotective action in trophic factor-deprived DRG neurons. In the present study, using immunocytochemistry, we have investigated some of the factors associated with apoptosis that are activated when nNOS activity is blocked with NOS inhibitor in DRG neurons in vitro. Marked elevation of bax was observed within a few hours of NOS inhibition in nNOS containing neurons, whereas pretreatment of cultures with l-arginine completely abolished this effect in almost all nNOS neurons and 8-bromo-cGMP in some neurons. The apoptosis precipitated by NOS inhibition was also partially prevented by a number of caspase inhibitors; of those a caspase-9 blocker was the most effective. These observations further support the neuroprotective role of NO/NO-cGMP in stressed DRG neurons in an autocrine fashion that involves the suppression of bax, caspase-3 and -9 activation.     

Dorsal root ganglion neurons of embryonic chicks contain nitric oxide synthase and respond to nitric oxide

We investigated the function of nitric oxide (NO) in dorsal root ganglion (DRG) neurons from 10 day embryonic chicks and adult birds. NADPH-diaphorase activity, a histochemical marker for nitric ooxide synthase (NOS) in paraformaldehyde-fixed neurons, and NOS-like immunoreactivity were localized in all neurons in thoracic and lumbar ganglia from embros. However, only a subset of neurons from adults contained NOS-like immunoreactivity and NADPH-diaophorase activity. Thus, embryonic chick DRG neurons have the potential to synthesize NO in response to elevated cytoplasmic Ca²⁺. We also investigated the ability of dissociated embryonic chick DRG neurons to respond to NO by examining the effects of NO donors and 8-bromoguasonine 3′,5′-cyclic monophosphate (8-Br-cGMP) on Ca²⁺ current (I_Ca) using the amphotericin-permeabilized patch-clamp technique: sodium nitroprusside (5 μM) reduced I_Ca to 0.68 ± 0.06 (mean±S.D., n = 5) of control, S-nitroso-N-acetylpenicillamine) (1 μM) reduced I_Cato0.44 ± 0.06 (n = 4) of control, while 8-Br-cGMP (1 mM) reduced I_{Cato0.58 ± 0.22 (n = 5)} of control. I_Ca was reduced in every neuron tested and this effect was partially reversed after ≈ 10min of washing. Thus, I_Ca of embryonic chick DRG neurons is inhibited by NO, possibly by a cGMP-dependent mechanism. These results indicate that all DRG neurons in embryonic chicks contain NOS-like immunoreactivity and respond to NO. Further, the percentage of NADPH-diaphorase positive neurons is reduced during development.     

Stimulation with N-methyl-D-aspartate or kainic acid increases cyclic guanosine monophosphate-like immunoreactivity in turtle retina: Involvement of nitric oxide synthase

In brain and retina, stimulation with excitatory amino acids (EAA) can generate nitric oxide (NO) and increase levels of cyclic guanosine monophosphate (cGMP). Because nitric oxide synthase (NOS) has been found in retinas of all species examined to date, an NO signal-transduction pathway is likely to be present in all retinas. We tested the hypothesis that stimulation of ionotropic glutamate receptors in turtle retina would result in increases in cGMP through an NOS/NO/cGMP pathway. Following in vitro incubations of turtle eye cups with the glutamate receptor agonists, N-methyl-D-aspartate (NMDA) or kainic acid (KA), we quantified the increases in cGMP-like immunoreactivity (cGMP-LI) by using enzyme-linked immunosorbant assay (ELISA) and localized the increased cGMP-LI by using an antibody against cGMP. Stimulation with NMDA or KA increased cGMP-LI in bipolar and amacrine cells as well as in some somata in the ganglion cell layer. Either KA or NMDA produced statistically significant increases in total retinal cGMP-LI by ELISA. To test the involvement of NO, we used the NOS inhibitors 7-nitroindazole and L-nitroarginine. Both inhibitors blocked virtually all of the KA- or NMDA-stimulated increases in cGMP-LI. These results indicate that activation of ionotropic glutamate receptors can increase cGMP in select retinal neurons. Differences between the agonist-evoked increases of retinal cGMP-LI suggest that there can be specificity in the activation of the NOS/NO/cGMP signal-transduction pathway by glutamate. This suggests that, in addition to short-term electrical changes, activation of ionotropic glutamate receptors also may produce longer term modulatory or metabolic effects involving NO/cGMP. J. Comp. Neurol. 404:75–85, 1999. © 1999 Wiley-Liss, Inc.     

Characterization of nitric oxide signaling pathways in the mouse retina

Nitric oxide (NO) is a gaseous neuromodulator with physiological functions in every retinal cell type. NO is synthesized by several nitric oxide synthases (NOS) and often functions through its second messenger, cyclic guanosine monophosphate (cGMP), and protein kinase G (PKG). This study combined NO imaging, immunocytochemistry, biochemistry, and molecular biology to localize NO and its downstream signaling pathways in the mouse retina. Neuronal NOS (nNOS) was localized primarily in puncta in the inner plexiform layer, in amacrine cells, and in somata in the ganglion cell layer. Endothelial NOS was in blood vessels. Light‐stimulated NO production imaged with diaminofluorescein was present in somata in the inner nuclear layer and in synaptic boutons in the inner plexiform layer. The downstream target of NO, soluble guanylate cyclase (sGC), was in somata in the inner and outer nuclear layers and in both plexiform layers. Cyclic GMP immunocytochemistry was used functionally to localize sGC that was activated by an NO donor in amacrine, bipolar, and ganglion cells. Cyclic GMP‐dependent protein kinase (PKG) Iα was found in bipolar cells, ganglion cells, and both plexiform layers, whereas PKG II was found in the outer plexiform layer, amacrine cells, and somata in the ganglion cell layer. This study shows that the NO/cGMP/PKG signaling pathway is functional and widely distributed in specific cell types in the outer and inner mouse retina. A better understanding of these signaling pathways in normal retina will provide a firm basis for targeting their roles in retinal pathology. J. Comp. Neurol. 520:4204–4217, 2012. © 2012 Wiley Periodicals, Inc.     

Development of tyrosine hydroxylase-like immunoreactive structures in the chick retina: three-dimensional analysis.

This study was designed to investigate the developmental profile of tyrosine hydroxylase-like immunoreactive structures in the chick retina in both frozen sections and wholemount preparations. In frozen sections, cells with tyrosine hydroxylase-like immunoreactivity were first detected in 10 to 15 cell rows from the innermost part of the inner nuclear layer on embryonic or incubation day 11. They were seen in the inner cell rows of the inner nuclear layer during later periods; by embryonic day 18, the immunoreactive cells were located 1 to 3 cell rows outward from the innermost part of the inner nuclear layer where mature immunoreactive cells mainly exist. The immunoreactive cells began to give rise to processes on embryonic day 13. The processes (possibly dendrites) gradually increased in number and intensity in sublayers 1 and 4 of the inner plexiform layer during prenatal life. Several days after hatching, an abrupt increase in immunoreactive processes was noted in sublayer 1 but not in sublayer 4. On the sixth postnatal day, retinal neural elements immunoreactive for tyrosine hydroxylase seemed to exhibit a distribution pattern similar to that of the adult chick. In wholemount retinas, immunoreactive cells were initially detected at the earliest stage of embryonic day 12 in a small circle termed "starting area" occupying the ventral part of the temporal retinal field. The closer to the "starting area," the earlier the retinal area began to express many immunoreactive cells. Thus tyrosine hydroxylase cell density in individual retinal areas, as represented by cell number per square millimeter, peaked in different developmental periods varying from embryonic day 12 to day 14. At this stage, immunoreactive cells were arranged irregularly in the retina. Thereafter, the cell density as well as total cell number gradually declined and reached a plateau around embryonic day 20 when tyrosine hydroxylase-like immunoreactive cells, like those in the mature retina, showed an even distribution throughout the retina.(ABSTRACT TRUNCATED AT 400 WORDS)