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.     

Differential expression of acetylcholinesterase molecular forms in neural retina and retinal pigmented epithelium during chick development

Neural retina (NR) and retinal pigmented epithelium (RPE) were used as a model for studying acetylcholinesterase (AChE) expression in neuronal and non-neuronal tissue during development. In chick embryo retina, increasing AChE activity appeared from day 7 to hatching, first in NR and then in RPE. NR contained 3 main AChE forms, 11.3S, 6.5S and 4.5S, resolved by sucrose density gradient centrifugation. An additional 19S form was exclusively detected in RPE whatever the extraction procedure followed. During differentiation, the proportions of AChE molecular form changed until they reached a steady state characteristics of mature neural retina, whereas in RPE, patterns of AChE molecular forms did not change significantly during development. Thus, 19S AChE appeared to be characteristic of non-neuronal retinal tissue. The expression of the AChE molecular forms in NR and RPE, and particularly the 19S form, was independent of the presence of the lens during retinal differentiation.     

Nitric oxide synthase-containing neurons in the hippocampus are preserved in trimethyltin intoxication

We studied the effects of trimethyltin (TMT) (9 mg/kg, p.o.) on the nitric oxide synthase (NOS)-containing neurons in the rat hippocampus by NADPH-diaphorase histochemistry and a biochemical assay of NOS activity. TMT exposure caused the typical behavioral changes and a loss of the CA3/4 pyramidal cells, which were NADPH diaphorase-negative. The scattered interneurons and the CA1 pyramidal cells, which were NADPH diaphorase-positive, were spared. Hippocampal NOS activity showed no reduction in the TMT-treated rats compared with the controls. These results provide evidence of the preservation of the NOS-containing neurons in TMT intoxication.     

Ageing has a differential effect on nitric oxide synthase-containing and catecholaminergic amacrine cells in the human and rat retina

In this study, we assessed the effects of normal ageing on the number, distribution, and somal area of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-positive (NADPHd⁺) and tyrosine hydroxylase-immunoreactive (TH-IR) amacrine cells in human and rat retina. By using a double-labelling immunohistochemical technique, we have shown that these two enzymes are located in separate amacrine cell populations in the human retina. In normal human retinas from organ donors, we have shown that there was no change in the number, somal area, or retinal distribution of NADPHd⁺ neurons over an age range of 19–89 years. In contrast, there was a significant decrease (P < 0.05) of 52% in the total number of TH-IR neurons in the group aged 65–89 years compared with the group aged 19–64 years. CA1 and CA2 TH-IR neurons were reduced by 44% and 55%, respectively. In young (3 months) and old (2 years) rats, the number of NADPHd⁺ neurons did not decrease with ageing, but the number of TH-IR neurons was significantly reduced by 21% (P < 0.05). In a companion study on monkey retina, we have shown that a postmortem delay of 12.5 hours between death and fixation results in a decrease of 33% in the number of both NADPHd⁺ and TH-IR neurons in the retina compared with the number in retinas fixed immediately after death. The findings of this study on the two subsets of amacrine cells, therefore, are likely to demonstrate the consequences of ageing in the retina and might contribute to visual impairment in the elderly. J. Comp. Neurol. 389:329–347, 1997. © 1997 Wiley-Liss, Inc.     

Cholinergic innervation of the smooth muscle cells in the choroid coat of the chick eye and its development

The mechanical and pharmacological characteristics of the cholinergic activation of the smooth muscle in the choroidal coat of the chick eye have been assessed in tissues isolated from birds 1 d posthatching using histological, electrophysiological, and immunological techniques. The choroidal coat is innervated by a dense network of cholinergic nerves that make en passant synapses with smooth muscle. Thirty-hertz stimulation of these nerves initiates red blood cell (RBC) movement in the vessels of the choroidal coat, and this activation is blocked by muscarinic ACh receptor (AChR) antagonists. Force-transducer recordings of nerve-induced contractions of this tissue have a slow onset and relaxation time course similar to those of smooth muscle contractions. Furthermore, since nearly half the cholinergic neurons innervating the choroid die within a defined period during development, the onset and pharmacology of this innervation were studied during embryogenesis. With a neural cytoskeletal-like immunostain, we demonstrated that choroid axons are present in peripheral tissue by stage (St) 29. Extracellular electrical recordings made after choroid nerve stimulation allowed us to distinguish axon from muscle responses. These procedures permitted us to examine the time course of the innervation of the smooth muscle. However, to visualize the postsynaptic smooth muscle response, it was necessary to treat the isolated preparation with tetraethylammonium chloride (TEA). Accordingly, TEA-enhanced electrical smooth muscle responses to single-nerve stimuli could be recorded only after St 39. Treatment of the nerve-muscle preparation with prostigmine allowed the recording of TEA-enhanced electrical activity as early as St 36 (1 d after the beginning of the normal choroid neuron death period). This synaptic activation was completely blocked by atropine or quinuclidinyl benzylate (QNB), and was not affected by alpha bungarotoxin (alpha BTX), indicating that, as in the posthatching tissue, neuromuscular transmission is mediated by muscarinic receptors. These results show that cholinergic muscarinic activation of the choroidal coat can occur as early as St 36, but that it is not as efficient as transmission later in embryogenesis.     

Growth hormone localization in the neural retina and retinal pigmented epithelium of embryonic chicks

It is well-established that growth hormone (GH) is present in the brain, spinal cord, and peripheral nerves of embryonic chicks, prior to the differentiation of pituitary somatotrophs, but its presence and distribution in retinal tissues is controversial. The possible presence of GH and GH mRNA in retinal tissues of early embryos has therefore been further evaluated. A 466-bp fragment of the pituitary GH cDNA, derived from a portion of exon 3 and spanning exons 4 and 5, was amplified by RT-PCR from reverse-transcribed mRNA from the pituitary glands of juvenile chicks and from the whole eye, neural retina, and retinal pigmented epithelium (RPE) of embryonic-day (ED) 9 chick embryos. In ED 9 embryos, GH immunoreactivity was demonstrated in the choroid and neural retina, in which it was particularly abundant in a layer of cells with the location and morphological appearance of retinal ganglion cells. GH immunoreactivity was also present in tissue sections of the RPE that were bleached to remove the melanin pigment. The intense GH staining in the RPE of ED 9 embryos was also revealed using a fluorescein-labeled GH antibody and confocal microscopy. At the ultrastructural level, GH detected by immunogold electron microscopy was present in the cytoplasm of RPE and neural retinal cells of ED 9 embryos. Although not associated with secretory granules, GH in the RPE was particularly associated with the membranes of the melanin granules. These results demonstrate that the neural retina and RPE are extrapituitary sites of GH production in early chick embryos, prior to the differentiation of the pituitary gland.     

Development of nitric oxide neurons in the chick embryo retina

Nitric oxide (NO) is a gas involved in neurotransmission in the central nervous system (CNS) and in vertebrate retinas. This paper describes five types of nitrergic neurons in developing and adult chick retina using the nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) reaction. Three of them, nitrergic types 1, 2 and 3, were observed in the inner nuclear layer, while nitrergic type 4 was observed in the ganglion cell layer; nitrergic type 5 were the retinal photoreceptors. Cell processes formed four nitrergic networks, which could be observed in the inner plexiform layer (IPL), at sublayers 1, 3a, 3b and 4. Another nitrergic network was observed in the outer plexiform layer (OPL). From hatching, the dendritic branches were completely developed in the IPL and in the OPL, forming the mentioned networks. Current evidence suggests that NO is coexpressed with other neurotransmitters in neurons of the CNS. Double-staining procedures, using NADPHd and 5HT immunohistochemistry in chicken retina, in a sequential or in an alternative manner, did not reveal the coexistence of these two neurotransmitters in the same neurons, but their networks matched in sublayers 1 and 4 of the IPL.     

Identification and localization of myosin superfamily members in fish retina and retinal pigmented epithelium

Myosins are cytoskeletal motors critical for generating the forces necessary for establishing cell structure and mediating actin‐dependent cell motility. In each cell type a multitude of myosins are expressed, each myosin contributing to aspects of morphogenesis, transport, or motility occurring in that cell type. To examine the roles of myosins in individual retinal cell types, we first used polymerase chain reaction (PCR) screening to identify myosins expressed in retina and retinal pigmented epithelium (RPE), followed by immunohistochemistry to examine the cellular and subcellular localizations of seven of these expressed myosins. In the myosin PCR screen of cDNA from striped bass retina and striped bass RPE, we amplified 17 distinct myosins from eight myosin classes from retinal cDNA and 11 distinct myosins from seven myosin classes from RPE cDNA. By using antibodies specific for myosins IIA, IIB, IIIA, IIIB, VI, VIIA, and IXB, we examined the localization patterns of these myosins in retinas and RPE of fish, and in isolated inner/outer segment fragments of green sunfish photoreceptors. Each of the myosins exhibited unique expression patterns in fish retina. Individual cell types expressed multiple myosin family members, some of which colocalized within a particular cell type. Because much is known about the functions and properties of these myosins from studies in other systems, their cellular and subcellular localization patterns in the retina help us understand which roles they might play in the vertebrate retina and RPE. J. Comp. Neurol. 513:209–223, 2009. © 2009 Wiley‐Liss, Inc.     

Neurogenesis of GABAergic cells in the chick retina

Two classes of retinal neurons in the chick retina, the horizontal and the amacrine cells, are GABAergic. This study evaluates the neurogenesis of glutamic acid decarboxylase immunoreactive cells in the chick retina. Twenty-five μCi [³H]thymidine was injected into eggs of 2–10 days and the embryos were sacrificed at embryonic day 18 (E18). Glutamic acid decarboxylase immunohistochemistry was revealed by avidin–biotin complex method followed by autoradiography of thymidine. We used the cumulative method for counting autoradiographic grains. At E3, 10% of the amacrine cells were thymidine negative/glutamic acid decarboxylase positive and this rate remained constant until E6. From E6 to E8 about 80% of the amacrine cells were thymidine negative/glutamic acid decarboxylase positive. At E9, 100% of these neurons had been generated. On the other hand, at E3 only 1.5% of the horizontal cells had been generated (thymidine negative/glutamic acid decarboxylase positive) while at E6 this number increased to 10%. From E6 to E9 the neurogenesis pattern was similar to that found for amacrine cells. Our data show that the great majority (80%) of glutamic acid decarboxylase positive amacrine and horizontal cells proliferate between E6 and E9, i.e. the last 3 days of the neurogenesis period. From E3 to E6 only 20% of the glutamic acid decarboxylase positive amacrine and horizontal cells are generated, which suggests that glutamic acid decarboxylase positive cells may require a specific signal at about E6, which triggers their withdrawal from the cell cycle.     

CGMP increases in satellite cells of nitric oxide synthase-containing sensory ganglia

cGMP and the enzymes, nitric oxide synthase (NOS) and heme oxygenase-1 (HO-1), the products of which stimulate soluble guanylyl cyclase activity, were investigated in cultured dorsal root ganglia (DRG), and nodose ganglia of adult rats. A dramatic increase of cGMP-positive satellite cells in ganglia cultured for 24 or 48 h was observed, particularly in Th8-L2 DRG and in nodose ganglia. These ganglia also contained most NOS-positive neurones, as reflected by NADPH-diaphorase histochemistry. HO-1 immunoreactivity increased in satellite cells, but in different cells to those in which cGMP increased. These results suggest that both NO and CO could be involved in signalling between neurones and satellite cells in sensory ganglia during regeneration.     

Distribution of ganglion cells in the retina of adult pigmented ferret

The retinal ganglion cell distribution in adult pigmented ferret was mapped in Nissl-stained retinas and in retinas back-filled with HRP after large bilateral injections of the enzyme into the brain. In common with other carnivores the ferret has an area of peak cell density equivalent to the area centralis and a prominent visual streak of high cell density extending horizontally across the retina. The maximum ganglion cell densities for the retinas were estimated to be 3500-5200 cells/mm2 in Nissl-stained, dehydrated retinas and 3300-4300 cells/mm2 in HRP-labelled, undehydrated retinas. Three cell types were distinguished in the HRP-labelled retinas and they appear to correspond to alpha-, beta- and gamma-cell types of cat retina. However, unlike in cat, the retinal ganglion cells of the ferret do not consistently fall into 3 distinct groups with respect to cell size, nor is there a tendency for the cells in the area centralis to be smaller than those in the peripheral retina. Estimates for the total number of ganglion cells of 82,000 and 88,000 were obtained from Nissl-stained retinas, and of 74,000, 75,000 and 78,000 from HRP-labelled retinas.     

Cell-by-cell reconstruction in reaggregates from neonatal gerbil retina begins from the inner retina and is promoted by retinal pigmented epithelium

For future retinal tissue engineering, it is essential to understand formation of retinal tissue in a 'cell-by-cell' manner, as can be best studied in retinal reaggregates. In avians, complete laminar spheres can be produced, with ganglion cells internally and photoreceptors at the surface; a similar degree of retinal reconstruction has not been achieved for mammals. Here, we have studied self-organizing potencies of retinal cells from neonatal gerbil retinae to form histotypic spheroids up to 15 days in culture (R-spheres). Shortly after reaggregation, a first sign of tissue organization was detected by use of an amacrine cell (AC)-specific calretinin (CR) antibody. These cells sorted out into small clusters and sent unipolar processes towards the centre of each cluster. Thereby, inner cell-free spaces developed into inner plexiform layer (IPL)-like areas with extended parallel CR(+) fibres. Occasionally, IPL areas merged to combine an 'inner half retina', whereby ganglion cells (GCs) occupied the outer sphere surface. This tendency was much improved in the presence of supernatants from retinal pigmented cells (RPE-spheres), e.g. cell organization and proliferation was much increased, and cell death shortened. As shown by several markers, a perfect outer ring was formed by GCs and displaced ACs, followed by a distinct IPL and 1-2 rows of ACs internally. The inner core of RPE spheres consisted of horizontal and possibly bipolar cells, while immunostaining and RT-PCR analysis proved that photoreceptors were absent. This shows that (1) mammalian retinal histogenesis in reaggregates can be brought to a hitherto unknown high level, (2) retinal tissue self-organizes from the level of the IPL, and (3) RPE factors promote formation of almost complete retinal spheres, however, their polarity was opposite to that found in respective avian spheroids.     

Nitric oxide stimulates gamma-aminobutyric acid release and inhibits glycine release in retina

Nitric oxide (NO) modulates the uptake and/or release of neurotransmitters through a variety of cellular mechanisms. However, the pharmacological and biochemical processes underlying these neurochemical effects of NO often remain unclear. In our study, we used immunocytochemical methods to study the effects of NO, cyclic guanosine monophosphate (cGMP), and peroxynitrite on the uptake and release of gamma-aminobutyric acid (GABA) and glycine in the turtle retina. In addition, we examined the involvement of glutamate receptors, calcium, and the GABA transporter in this GABA uptake and release. We also tested for interactions between the GABAergic and glycinergic systems. In general, we show that NO stimulated GABA release and inhibited glycine release. The NO-stimulated GABA release involved calcium-dependent or calcium-independent synaptic release or reversal of the GABA transporter. Some effects of NO on GABA release involved glutamate, cGMP, or peroxynitrite. NO promoted glycine uptake and inhibited its release, and this inhibition of glycine release was influenced by GABAergic modulation. These findings indicate that NO modulates the levels of the inhibitory transmitters GABA and glycine through several specific biochemical mechanisms in different retinal cell types and layers. Thus it appears that some of the previously described reciprocal interactions between GABA and glycine in the retina function through specific NO signaling pathways.     

Monocyte-derived IL-10-secreting dendritic cells in choroid plexus epithelium

Choroid plexuses form an interface between peripheral blood and cerebrospinal fluid. Dendritic-like cells have been reported in a few studies of choroid plexuses in man. Here we used electron microscopy and immunophenotyping to precise the morphologic features and phenotype of these cells. Examination of 10 human choroid plexuses evidenced intra-epithelial dendritic cells with a clear cytoplasm, reniform nucleus and long expansions. These cells express MHC Class II, CD11b, CD14, CD32, CD68 and IL-10, but not CD40, CD80 or CD86, suggesting an immunosuppressive role for these dendritic cells. Their sentinel position could make them participate to the immunological silence of the brain.     

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.     

Nitric oxide synthase in learning-relevant nuclei of the chick brain: Morphology,distribution, and relation to transmitter phenotypes

Nitric oxide (NO) has been implicated in learning in the hatchling chicken. To examine morphological and neurochemical properties of neurons that contain NO synthase (NOS) in brain regions known to be involved in learning and memory, the NADPH-diaphorase technique was used in conjunction with immunocytochemistry and tract tracing. A distinct cell type was NOS-labeled in the lobus parolfactorius (LPO) in the telencephalon, and neurons were labeled in the area ventralis of Tsai (AVT), the substantia nigra (nucleus tegmenti pedunculo-pontinus, pars compacta, TPc), and the locus coeruleus in the brainstem. Thus, NO may influence processes of learning and memory in the forebrain after release from intrinsic neurons and/or from extrinsic NOS-projections originating from the brainstem. DiI-tracing revealed that most of the NOS-positive neurons in the AVT/TPc project to the basal forebrain. The majority of tyrosine hydroxylase-positive (presumptive dopaminergic) neurons in the AVT and TPc expressed NOS. Double-labeling with antibodies to tyrosine hydroxylase, choline acetyltransferase, somatostatin, and the neurotrophin receptor as a marker for noradrenergic coeruleus neurons showed that NOS was not colocalized with noradrenergic or somatostatinergic neurons, and that less than a third of the cholinergic neurons were double-labeled for NOS. Injections of 6-hydroxydopamine into the brainstem did not reduce the density of NOS-labeled fibers in the LPO, indicating that most of the NO in the LPO originates from intrinsic neurons in the basal forebrain. Thus, NOS-containing presumptive local circuit neurons in the LPO are the most likely source of NO involved in learning of passive avoidance tasks in hatchling chicks. J. Comp. Neurol. 383:135–152, 1997. © 1997 Wiley-Liss Inc.     

Expression of axonin-1 in developing amacrine cells in the chick retina

This study focused on the temporal and spatial pattern of expression of the cell adhesion molecule axonin-1 in amacrine cells and the identification of these cells in the developing chick retina. We analyzed 5-20-day-old chick embryos. The antigen was localized and visualized by the indirect immunogold and the immunofluorescence technique. Colocalization studies with antibodies against tyrosine hydroxylase, acetylcholinesterase, choline acetyltransferase, parvalbumin, calbindin, and calretinin served to characterize these cells further and to explore whether they have other properties in common. Axonin-1 was expressed in amacrine cells from E8 onward in the inner nuclear, in the inner plexiform, and in the ganglion cell layer. Their maturation showed a gradient similar to that found for amacrinogenesis. Expression was closely correlated with the period when the cells develop and shape their processes. The interneurons were classified with reference to Cajal, and most of the morphological types described by him were found. In addition, some cells were considered as axon-bearing amacrine cells. However, the total number of labeled cells was rather small. At least two morphologically different types terminated in each of the inner plexiform sublayers. Narrow- and wide-field arbors indicated the existence of a diversified network. The colocalization studies revealed that the neurotransmitters and neuropeptides overlapped partially with axonin-1 expression. This indicated that axonin-1-immunoreactive amacrine cells were also functionally diverse.     

GABA content in the retina of pigmented and albino rats

Reduction of the melanin precursor DOPA associated with albinism leads to spatiotemporal disturbances in retinal neurogenesis and thus seems to be responsible for numerous neuronal alterations found in albino retinae. To investigate whether these cellular alterations are reflected in retinal neurotransmitter concentrations we compared the levels of GABA and glutamate in the retina of adult pigmented Long Evans and albino Wistar rats using reversed phase-liquid chromatography (RP-HPLC). When normalized to retinal weight, GABA levels showed a statistically insignificant trend to be lower and glutamate values to be higher in albinos than in pigmented animals. The ratio of glutamate to GABA was significantly higher in albino than in pigmented retinae. As numerous studies have shown that the balance between GABA and glutamate plays a crucial role for establishing direction selectivity, these results are discussed in relation to direction selectivity and defects in the optokinetic system of albinos.