Endothelial Nitric Oxide Synthase Is Expressed in Amacrine Cells of Developing Human Retinas

Abstract

purpose. To examine the expression and cellular distribution pattern of endothelial nitric oxide synthase (eNOS) in the developing human retina and to compare its expression with that in rats.

methods. Expression of eNOS was examined by immunohistochemistry in retinas of humans ranging from 8.5 to 28 weeks of gestation (WG) and of rats.

results. In the developing human retina, eNOS expression was first detected in the proximal margin of the neuroblastic layer in the incipient fovea-surrounding area at 12 WG. At 17 to 28 WG, eNOS-immunoreactive cells were located in the innermost part of the inner nuclear layer and in the ganglion cell layer, expanding to both temporal and nasal retinas and the processes projecting into the inner plexiform layer. These eNOS-positive cells coexpressed syntaxin and glutamate decarboxylase, and are probably GABAergic amacrine cells. The onset of eNOS expression in developing amacrine cells, however, preceded the invasion of retinal vasculature, long before vascular function involving these cells can be expected, suggesting that eNOS has a role not only in vasoregulation but also in retinal development. From 20 WG on, eNOS was also detected in the photoreceptors adjacent to the fovea. eNOS expression in amacrine cells and photoreceptors was observed in the central-to-peripheral and temporal-to-nasal gradients. However, in the developing rat retina, eNOS was expressed exclusively in the vascular endothelial cells.

conclusions. The results support that eNOS plays a role, not only in the regulation of vascular function but also in the process of retinal development in humans.

In addition to its well-known role in regulating cardiovascular functions, nitric oxide (NO) has been implicated in a variety of physiological and pathophysiological processes in the nervous system, such as neurotransmission, brain development, synaptic plasticity, trophic function, and glutamate-mediated toxicity. ^{1 2 3 4 5} NO is a highly diffusible and short-lived signaling molecule that is produced on demand by activation of nitric oxide synthase (NOS). Unlike other intercellular messenger molecules that are regulated by a series of processes including synthesis, posttranslational modification, formation of synaptic vesicles, and regulation of release, the production of NO is mainly regulated by a class of NOSs that catalyze the conversion of l-arginine and oxygen to l-citrulline and NO. Of the three NOS isoforms—neuronal (n)NOS, endothelial (e)NOS, and inducible (i)NOS—eNOS originally occurs discretely in vascular endothelial cells and is mostly associated with the potent vasodilator property of NO. ⁶  

eNOS expression is not only found in vascular endothelial cells, but also in neurons in selected regions of the central nervous system (CNS). ^{7 8 9} eNOS in the pyramidal cells of the hippocampus has been shown to participate in the induction of long-term potentiation, ⁹ a form of synaptic plasticity. eNOS is expressed in neural stem and precursor cells and has been implicated in the regulation of neuronal progenitor cell proliferation and differentiation. ¹⁰ Astrocytes in the CNS respond to viral infection by an increase in eNOS expression. ¹¹ These observations have indicated that eNOS has important biological functions in the nervous system, in addition to its role affecting vascular functions. 

As in other tissues, eNOS has been localized to endothelial cells lining the vasculature in the retina. ^{12 13} In addition to the presence of eNOS in the blood vessels, a recent study has demonstrated that cells in the ganglion cell layer (GCL) can be induced to express eNOS after ischemia–reperfusion injury. ¹² Moreover, eNOS immunoreactivity (IR) was detected in retinal ganglion cells (RGCs) in the postnatal rat retina. ¹⁴ Photoreceptors, amacrine cells, and RGCs in the developing chick retina have also been shown to contain basal levels of eNOS. ¹⁵ However, the expression of eNOS in the developing human retina is still unknown, and consequently, so is the role that eNOS may have in the development of human retina. Therefore, the purpose of the present study was to examine the expression and cellular distribution pattern of eNOS in the developing human retina, and thereby help to elucidate the possible role of eNOS in retinal development. The expression pattern of eNOS in the developing human retina is also compared with that of the rat retina. 

Methods

Collection and Preparation of Human Fetal Tissues

Ten human fetal eyes, ranging in age from 8.5 to 28 weeks of gestation (WG), were obtained under approved protocols, and all study procedures were in accordance with the guidelines set forth in the Declaration of Helsinki and the terms of all relevant local legislation. The age of the fetus was determined by a combination of medical records, supersonic check, and crown–rump length. Fetuses older than 17 WG were perfused through the left ventricle with 0.9% saline, followed by 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS, pH 7.4) at 4°C. The eyes were then quickly enucleated; cornea, lens, and vitreous body were removed; and the eyecups were postfixed in the same fixative for a further 24 to 48 hours. For fetuses at 12 to 17 WG, the eyecups were immersed in the same fixative for 1 to several days. For fetuses younger than 12 WG, whole eyes were fixed for several days. The tissue was cryoprotected by immersion in 30% sucrose at 4°C overnight, serially sectioned at 10 to 20 μm on a cryostat, mounted on slides, and stored at −70°C. 

Collection and Preparation of Rat Tissues

All procedures for the care and handling of animals were approved by the University of Hong Kong’s Committee on the Use of Live Animals in Teaching and Research and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Retinas from Sprague-Dawley rats were obtained at embryonic day (E)18, and postnatal days (P)0, P7, and P14 and from adult (at least three animals at each stage except only one animal at E18). Adult rats were anesthetized with 7% chloral hydrate (42 mg/100 g body weight, intraperitoneal injection) and perfused transcardially with saline followed by 4% paraformaldehyde. Eyes were removed and postfixed in the same solution for 2 hours at 4°C. The postnatal rats were decapitated and eyeballs were removed. Eyecups were immersed in fresh fixative for 2 hours or overnight at 4°C and then left in 30% sucrose at 4°C overnight, and 10-μm-thick cryosections were cut. 

Immunohistochemistry

A rabbit polyclonal antibody raised against the C-terminal of human eNOS was used (cat. no. sc-654; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). A rabbit polyclonal antibody raised against the C-terminal of rat nNOS was used as a control (cat. no. sc-648; Santa Cruz Biotechnology, Inc.). The peptides, which the eNOS antibody and the nNOS antibody were raised against, respectively, were used for the preabsorption of the antibodies (cat. no. sc-654p and sc-648p; Santa Cruz Biotechnology, Inc.). Other antibodies used in the study included a mouse monoclonal antibody against CD34 (cat. no. 07-3403; Zymed, South San Francisco, CA), to identify vascular endothelial cells; a mouse monoclonal antibody against ki-67 (cat. no. 18-0192; Zymed), to identify proliferating cells; a mouse monoclonal antibody against NeuN (cat. no. MAB377; Chemicon, Temecula, CA), used as a neuronal marker; a mouse monoclonal antibody against pan neurofilament (cat. no. 18-0171; Zymed); a mouse monoclonal antibody against β-tubulin III (cat. no. T8660; Sigma-Aldrich, St. Louis, MO), to identify RGCs; a mouse monoclonal antibody against syntaxin 1A isoform (HPC-1; cat. no. S0664; Sigma-Aldrich), to identify amacrine cells and horizontal cells, although it also labels RGC axons in the nerve fiber layer during development; a rabbit polyclonal antibody against glutamate decarboxylase isoform GAD67 (a gift from Jang-Yen Wu, Departments of Molecular Biosciences and Medicinal Chemistry, University of Kansas), ¹⁶ to identify GABAergic neurons; a mouse monoclonal antibody against 7G6 (a gift from Peter MacLeish, Neuroscience Institute, Morehouse School of Medicine, Atlanta, GA), ¹⁷ to identify cone photoreceptors; and a mouse monoclonal antibody against rhodopsin (cat. no. O4886; Sigma-Aldrich), to identify rod photoreceptors. 

Sections were blocked with 10% normal goat serum and 1% bovine serum albumin in PBS containing 0.3% Triton X-100 and 0.1% sodium azide and then incubated at 4°C overnight with eNOS antibody (1:300). Specific binding was detected with an immunoperoxidase protocol (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA) and developed with 3,3′-diaminobenzidine (DAB) in 0.1 M acetic buffer (pH 6.5) containing 1% ammonium nickel sulfate, 0.004% ammonium chloride, and 0.0005% H₂O₂. For double-labeled immunofluorescent experiments, eNOS antibody was detected with an amplification kit (Alexa Fluor 568-Tyramide Signal Amplification Kit; Invitrogen, Carlsbad, CA). The sections were then incubated with antibodies for a second layer of labeling and then with Alexa Fluor 488–conjugated donkey anti-mouse IgG or anti-rabbit IgG. The slides were mounted in antifade mounting medium (Dako Corp., Carpinteria, CA) and observed under a confocal microscope (Bio-Rad Laboratories, Hercules, CA). Immunostaining of ki-67 was performed as already described, except before incubation of primary antibody; sections were heated in 0.01 M citrate buffer (pH 6.0) containing 0.3% Triton X-100 for 20 minutes at 85°C and then cooled for 30 minutes at room temperature. Control experiments included: primary antibody preabsorbed with a 20-fold excess of a specific peptide mapping to the C-terminal of human eNOS, which the eNOS antibody was raised against (Santa Cruz Biotechnology, Inc.) or a peptide mapping to the C-terminal of rat nNOS, which the nNOS antibody was raised against, from the same company. 

Results

Expression of eNOS in Developing Human Retinal Cells

In the developing human retina, eNOS immunostaining was generally detected in endothelial cells of the blood vessels located on the vitreous surface (Fig. 1A) , between the nerve fiber layer (NFL) and GCL (Fig. 1D) , and in the choroid, from 10 to 28 WG (Fig. 1) . At 12 WG, despite strong staining associated with the blood vessels of the vitreous and the choroid, a few weak eNOS-stained cells were detected in the inner border of the neuroblastic layer (NBL; Fig. 1B ) near the optic pole, adjoining the incipient fovea-surrounding area, where differentiation first begins during retinal development (Li X, Yip HK, unpublished data, 2005, and previous studies ^{18 19 20 21} ). From 17 WG onward, strong eNOS expression was found in cells located in the proximal inner nuclear layer (INL; probably corresponding to amacrine cells) and in the GCL (probably corresponding to ganglion cells or displaced amacrine cells; Figs. 1C 1D ). The eNOS-containing cells had round-to-ellipsoid profiles and emitted processes toward the inner plexiform layer (IPL; Figs. 1C 1D ). However, no elaborate arborization was detected in the IPL. Occasional short secondary branches extended from the thick primary process (Figs. 1C 2A) . The ratio of the number of eNOS-labeled cells in the GCL and the INL was ∼1 to 3 at 17 WG. 

We performed two stringent control experiments, to verify the specificity of the anti-eNOS antibody used in the study: (1) Preabsorption of eNOS antibody with an excess amount of eNOS-blocking peptide (the C-terminal peptide of human eNOS used as the immunogen to raise the eNOS antibody) completely eliminated immunolabeling, not only in the retinal cells, but also in the blood vessels located in the vitreous, retina, optic nerve head, and choroid (Figs. 2B 2D 2F) ; (2) peptide mapping to the C-terminal of rat nNOS, from the same company where the eNOS antibody was purchased (Santa Cruz Biotechnology, Inc.), did not eliminate the eNOS-IR in both the retinal cells and vasculature (Fig. 2C) . The labeling pattern was similar to the eNOS-staining without any preabsorption. Furthermore, preabsorption of nNOS antibody with peptide mapping to the C-terminal of eNOS did not eliminate the immunostaining of a subset of nNOS-IR amacrine cells. It has also been demonstrated that nNOS-IR amacrine cells and their processes have a close association with the retinal vessels and appear much later in the developing human retina, at 28 WG (data not shown). ²² However, we did not find a similar association of eNOS-IR nonvascular retinal cells and their processes with the vasculature. Therefore, the eNOS-IR nonvascular retinal cells detected in this study are not the same as the nNOS-IR amacrine cells previously described. ²²  

In a 16-WG flatmounted retina, eNOS expression was essentially confined to cells in the area surrounding the incipient fovea (Fig. 3B) . At this point in development, strong eNOS expression associated with the developing vasculature was restricted in two vascular fronds emanating from the superior and inferior poles of the optic disc (Fig. 3A) . Thus, it is most likely that eNOS is expressed by retinal cells in the intermediate location of the incipient fovea and not by endothelial cells associated with the developing retinal vasculature. 

More eNOS-positive cells were observed in the temporal half at 17 WG—mainly accumulating in the incipient fovea-surrounding area—than in the nasal half of the retina in a horizontal section (Fig. 4) . Thus, eNOS expression displayed a temporal-to-nasal and a central-to-peripheral gradient. This spatial expression pattern was in line with reports on the differentiation pattern of retinal neurons in the developing human retina (Li X, Yip HK, unpublished data, 2005, and a previous study ²¹ ). At this stage, eNOS expression in the developing vessels was still restricted to a small area surrounding the optic disc, to the optic nerve, and to the choroid. 

From 17 to 28 WG, even with the invasion of eNOS-stained blood vessels, there was no close spatial relationship of the eNOS-IR cells in the GCL and INL to the retinal vasculature. Colocalization studies with CD34, the cell marker of vascular endothelial cells, found that eNOS-labeled cell bodies, closely apposed to the retinal vasculature, overlapped extensively with CD34-IR. However, there was no CD34 coexpression in the eNOS-labeled retinal cells in the GCL and INL (Fig. 5) . To confirm that the eNOS-expressing retinal cells are postmitotic neurons, we performed a double-labeling experiment with Ki-67, a cell marker for mitotically active cells (Fig. 6) . We found that eNOS-positive retinal cells did not express Ki-67, indicating that eNOS was expressed in differentiating retinal cells and not in actively proliferating cells. These eNOS-IR retinal cells also displayed morphologic characteristics consistent with the relatively mature neurons that have a round or oval cell soma and nucleus and branching processes. 

In the rat retina, at the age of E18 to adult, eNOS-immunolabeled cells were detected only in the perivascular cells of blood vessels, and expression was absent in the retinal neurons. At P0 and P7 (Figs. 7A 7B) , eNOS-IR vessels were found on the vitreous surface of the retina. At P14 and in adult eyes, labeled vessels began to appear in deeper layers in the proximal and distal INL and extended radially through the retina (Figs. 7C 7D) . The labeling intensity of retinal vessels decreased from P14 to adult, suggesting that eNOS expression may be more related to development of vasculature in the rat retina. All vessels in the choroid were also eNOS positive (Figs. 7A 7B 7C) . The results obtained in the present study on eNOS expression in the developing rat retina are in good agreement with those previously reported for the localization of eNOS-IR in rat retina ¹² and also in accordance with previous findings on the development of retinal vessels in the rat. ²³  

Identity of eNOS-Immunoreactive Retinal Cells

To determine the identity of eNOS-IR cells, markers of specific classes of retinal neurons were used in the double-labeled immunofluorescence experiments with eNOS. We found that eNOS-positive cells in the GCL and INL adjoining the IPL were colocalized with NeuN, indicating that these cells are neurons (Fig. 8A) . However, they were not coexpressed with neurofilament (Fig. 4)and β-tubulin III (Fig. 8B) , specific cell markers for RGCs, suggesting that eNOS-stained cells in the GCL or INL were not RGCs. Coexpression of eNOS with syntaxin, a general marker of amacrine cells, is evident in the cells in the GCL and INL (Fig. 8C) . Thus, eNOS expression identified syntaxin-expressing amacrine cells and displaced amacrine cells in the INL and GCL, respectively. Furthermore, we sought to determine whether eNOS expression identifies amacrine cells expressing the glutamate decarboxylase (GAD) 67-kDa isoform (Fig. 8D) . Extensive colocalization of eNOS with GAD67 occurred in this population of amacrine cells, indicating that eNOS-expressing amacrine cells in the INL and GCL are GABAergic amacrine cells. 

eNOS Expression in Photoreceptors

From 20 WG on, eNOS expression was also detected in some photoreceptors (Figs. 8A 8D) . Scattered eNOS-IR in the photoreceptor layer was observed first in the incipient fovea-surrounding area and gradually extended into a larger area as development progressed. Some of the eNOS-positive photoreceptors expressed 7G6, a marker for cone photoreceptors (Fig. 9) . Coexpression of eNOS and rhodopsin, a rod photoreceptor marker, was not confirmed, probably because of the limited number of rhodopsin-positive rod photoreceptors detected at this developmental stage and the poor integrity of the outer retinas in the specimens at 28 WG (data not shown). Thus, we cannot rule out the possibility that some of the eNOS-labeled photoreceptors are rod photoreceptors. 

Discussion

Two lines of evidence in recent studies have demonstrated the dynamic expression pattern of different NOS isoforms in the CNS. First, knockout mice carrying targeted mutations in the nNOS genes display residual NOS in the brain. ²⁴ Second, eNOS, once thought to be present only in vascular endothelial cells, has now been found to be expressed by neurons and glial cells in the CNS. ^{7 8 9} These results suggest that eNOS plays a role in neural functions, in addition to the regulation of vascular activities. In this study, we provide the first evidence showing that, in addition to vascular endothelial cells, eNOS is expressed in a small population of retinal cells in the developing human retina. Our immunohistochemical data demonstrate that eNOS may have a dual role in both vascular and neuronal development of the human fetal retina. 

Except for the demonstration of eNOS expression in retinal neurons, our findings on the expression pattern of eNOS in the developing human retinal vasculature are in general agreement with previous observations. ^{25 26} When eNOS-labeled amacrine cells were first detected in the incipient fovea-surrounding area at 12 WG, eNOS expression associated with retinal vasculature was restricted to the optic nerves. As development proceeded, when eNOS expression in amacrine cells had spread to the temporal and nasal retina, retinal vessels were still primarily localized to the area surrounding the optic disc, mainly at the superior and inferior retina. The fact that eNOS-expressing cells coexpressed NeuN but not CD34, a vascular endothelial cell marker, suggests that eNOS is expressed in retinal neurons and not in vascular endothelial cells during early fetal development. The results revealed that the onset of eNOS expression in developing amacrine cells preceded the invasion of retinal vasculature in human fetal retina, indicating that eNOS may play a role in retinal development. Furthermore, the continued expression of eNOS in syntaxin-positive cells and the subsequent increase of eNOS-expressing amacrine cells in late development suggests that eNOS plays a more prominent role in terminal differentiation and/or cell maintenance rather than in initial differentiation of amacrine cells. The correlation of eNOS with GAD67 expression in amacrine cells indicated that GABAergic amacrine cells may also require eNOS expression for terminal differentiation and maintenance of the GABAergic phenotype. The absence of expression of the proliferation marker Ki-67 in eNOS-containing amacrine cells suggests that expression of eNOS occurs only in postmitotic cells in the developing retina. These findings are consistent with the observations that NO may be essential in the differentiation of neural precursor cells during neurogenesis. ²⁷ Perhaps one of the physiological roles of eNOS in developing retina is to contribute to the terminal differentiation and maintenance of amacrine cells through the release of NO. Further studies are necessary to test this hypothesis. 

There are some disagreements about the cellular location of eNOS in the retina. Cheon et al. ¹² and Ju et al. ¹³ demonstrated that eNOS-IR could only be detected in retinal vessels, but not in neurons, in the normal rat retina. These findings are consistent with our observations in the rat retina. Only after ischemia–reperfusion injury could eNOS expression be induced in the cells of the GCL. ¹² Other investigators have also detected eNOS mRNA in cultured RGCs and amacrine cells. ²⁸ In contrast, Tsumamoto et al. ¹⁴ reported that all RGCs in the normal postnatal rat retina immunohistochemically express eNOS protein and that NO can be detected in cultured RGCs. In their studies, however, eNOS-IR in the retinal vasculature, which should be present at the age they investigated, was not demonstrated. Goureau et al. ¹⁵ localized eNOS-IR in the photoreceptors, amacrine cells, and RGCs of the developing chick retinas. Differences in sensitivity between the antisera, differences in experimental conditions, or differences in species perhaps explains the discrepancy. 

In the present study, we used the most stringent controls in immunohistochemistry to verify the specificity of the eNOS antibody, by performing the preabsorption with a peptide specific to the eNOS antibody, or with a peptide specific to the distinct sequence of nNOS, which is the closest related molecule to eNOS in structure and function. Preabsorption of the eNOS antibody with eNOS peptides but not peptide specific to nNOS, completely abolished eNOS-IR. Furthermore, the eNOS antibody used in this study intensively labels blood vessels on the vitreous surface, in the retina, the choroid, and the optic nerve head of both human and rat tissues. In addition, the spatiotemporal pattern of eNOS expression in the developing human retinal vasculature in our study conformed completely with observations in previous studies. ^{25 26} The results from the control experiments established the validity of our immunohistochemical data. 

We demonstrated that, at 17 WG, eNOS-IR was primarily localized in amacrine cells in the INL and displaced amacrine cells in the GCL and processes in the inner and outer plexiform layer (OPL) at the incipient fovea-surrounding area and eNOS-IR spread peripherally with increasing age (28 WG was the latest gestation age examined). The appearance of eNOS-expressing cells coincided with synaptogenesis in the IPL and the proportion of these cells increased concomitantly with synaptic maturation, consistent with previous findings in humans. In the developing human retina, eNOS expression was observed in a temporal-to-nasal and central-to-peripheral gradient, confirming the sequences of retinal maturation in many species, including humans. ^{21 29 30} eNOS-IR photoreceptors, with immunolabeled cell bodies in the ONL and synaptic formation in the OPL, appeared in a similar manner and proceeded in a temporonasal and central peripheral sequence. On the whole, our results suggest that a wave of eNOS-positive cells appears at the incipient fovea at ∼17 WG and advances in a central–peripheral pattern across the retina as development progresses. Significantly, our results also suggest that this wave of eNOS-expressing cells is coincident with a central–peripheral pattern of synaptic formation in the IPL and OPL. This strongly suggests that eNOS expression in the INL, GCL, and ONL is associated with the formation of synaptic contacts by amacrine cells, displaced amacrine cells, and photoreceptors in the IPL and OPL, respectively. It has been shown that NO mediates the refinement of visual projections during development. ³¹ In addition, NO appeared to participate in learning and hippocampal synaptic plasticity. ^{8 9} It is plausible that eNOS and NO in the retinal cells also function as retrograde messengers in the refinement of synaptic connection and modulate neuronal transmission from photoreceptors to ganglion cells in the developing human retina. In this study, eNOS-labeled cells, apart from the endothelium of the blood vessels, can be found only in human retina, not in rat retina. There may be species variation in eNOS expression in the developing retina. 

Expression of NOS in the photoreceptors is still controversial. Immunochemical and NADPH-diaphorase (NADPHd) histochemical staining failed to detect NOS in photoreceptors. Other investigators have localized NOS in bovine photoreceptor inner segments. In contrast, NOS activity was said to be present in the outer segments in another report. Our study revealed the expression of eNOS in a small quantity of photoreceptors expressing 7G6 in the ONL, suggesting that eNOS expression may identify early cone photoreceptors. NADPHd reactivity has also been reported in a subpopulation of cone photoreceptors in adult human retina, possibly representing the blue cones. Furthermore, NADPHd histochemistry also identified the short-wavelength-sensitive (SWS or blue-sensitive) cone in the cone-dominated retina of the tree shrew. ^{32 33 34 35 36} Thus, the eNOS-IR photoreceptors detected in this study may contribute to the NOS activity detected with NADPHd histochemistry in the previous studies. These findings are consistent with the morphologic characteristics and expression pattern described for the SWS (S cones) in human fetal retina. Colocalization experiments are needed to elucidate further the identity of the eNOS-expressing cones. ²⁰  

This is the first report to describe the presence of eNOS in subsets of neurons in the developing human retina. The spatial and temporal sequence of eNOS expression correlates with the period of amacrine cells and photoreceptor differentiation and synaptogenesis, consistent with a role for eNOS in retinal development, in addition to the regulation of retinal circulation. NO acts as a modulator of neuronal transmission in the mature nervous system, including the retina. ^{37 38} Recent studies have suggested that in addition to their roles in synaptic communication in the mature brain, neurotransmitters and neuromodulators have a trophic role in neuronal maturation at an early developmental stage. Thus, a neurotransmitter–neuromodulator can take multiple forms and exert several actions at different developmental stages. ^{39 40} Therefore, it is conceivable that NO produced by retinal cells behaves in a similar manner. It is, however, thus far unclear how NO participates in neuronal development. It has been proposed as a retrograde messenger that acts downstream of the N-methyl-d-aspartate (NMDA) receptor on presynaptic terminals during development. ^{3 41} It has been suggested that NO is important in the regulation of neuronal progenitor cell proliferation, migration, differentiation, and neurite outgrowth ^{42 43} and in brain plasticity. ⁴² Furthermore, the expression of NOS may be crucial to the establishment of the appropriate pattern of synaptic connections in the visual target. ^{31 44} Our results showing the presence of eNOS expression in the amacrine and cone photoreceptor cells raised the possibility that NO, released by these cells and their processes in the extracellular space, could act as a diffusible tropic factor for the growing axons to make synaptic contacts, or could serve as a retrograde signal in the shaping of terminal arbors to match their dendritic targets. Therefore, a diffuse signal like NO could play a central role in the formation and stable maintenance of synapses during retinal development. 

Our data demonstrate that eNOS is present predominantly in subpopulations of GABAergic amacrine cells. GABA and NOS have been colocalized in the brain. ^{45 46} Previous studies have shown that the vertical glutamatergic flow of visual information from photoreceptors to ganglion cells is modulated by GABAergic inputs from horizontal and amacrine cells in the ONL and INL, respectively. ^{47 48} In this respect, it is important to note that GABAergic amacrine cells receive glutamatergic input from bipolar cells. These bipolar cells, in turn, are modulated by negative feedback from GABAergic amacrine cells. ⁴⁹ Thus, interactions between GABAergic and glutamatergic systems are functionally important in establishing a coherent micronetwork in the retina, and eNOS could be involved in this synaptogenesis. In addition to GABA, it has been demonstrated that the NMDA receptor is localized in NOS neurons in the retina. ⁵⁰ The colocalization of NOS and NMDA receptors suggests that NOS-expressing amacrine cells participate in glutamatergic circuits in the retina and that NOS may be triggered by the activation of the NMDA receptor. The increase of intracellular calcium, mediated by both L-type Ca²⁺ channels via GABA receptors and by ligand-gated Ca²⁺ channels by NMDA receptors, is believed to be involved in a variety of developmental events in CNS, such as neuronal migration ⁵¹ and neurite outgrowth. ^{52 53} It is reasonable to speculate that mobilization of intracellular Ca²⁺ ions by both types of channels plays a role in activating eNOS in developing retinal cells and that the spatiotemporal mobilization of the intracellular Ca²⁺ mediated by different signaling pathways could influence eNOS activity in a variety of developmental events in the retina. 

A significant finding in our studies was the apparent association of eNOS expression with the development of retinal neurons. Whether there is indeed a causal relationship between these two phenomena cannot be concluded from our data. However, our data suggest that with the prenatal detection of eNOS expression in human fetal retina and with the newly discovered role of neurotransmitters–modulators as early signaling molecules for CNS development, the potential involvement of eNOS in neuronal development and differentiation should be considered to be one of the possible functions of the eNOS/NO system. 

 Supported by grants from the Committee on Research and Conference Grant, the University of Hong Kong and the Regional Grant Council of Hong Kong.

 Submitted for publication October 9, 2004; revised August 30 and November 8, 2005; accepted March 13, 2006.

 Disclosure: S. Li, None; D. Tay, None; S. Shu, None; X. Bao, None; Y. Wu, None; X. Wang, None; H.K. Yip, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Expression of eNOS during human retinal development. The expression of eNOS in retinal sections was characterized by immunohistochemistry at different stages of prenatal retinal development from 10 to 28 WG. (A) At 10 WG, eNOS expression was detected in the cells of the blood vessels on the vitreous surface of the retina ( ) and in the choroid only. (B) At 12 WG, in addition to the eNOS-stained vessels, a few weak eNOS-staining cells are detected in the proximal NBL (arrows). eNOS-positive cells (arrows) were seen in the GCL and proximal margin of INL at 17 (C) and 28 (D) WG. Immunoreactive processes (arrowheads) can be seen projecting from these cells toward the IPL. (C) Secondary branch from the primary process of the eNOS-IR cells in the proximal INL. (D) Note that a retinal vessel (open arrowhead) at the GCL also displayed eNOS-IR. (C, D, insets) Higher magnification of the boxed regions. Scale bar, 50 μm.

Figure 2.

 

Specificity of eNOS immunoreactivity (IR) in developing human retina. Sections were immunostained with eNOS antibody without preabsorption (A, E); with preabsorption by eNOS C-terminal peptide, which eNOS antibody was raised against (B, D, F); with preabsorption by a peptide mapping to C-terminal of nNOS from the same company (C). At 17 WG, eNOS-IR cells (arrows) were seen in the GCL and proximal INL, with processes (arrowheads) projecting into the IPL and the choroid (A); preabsorption with eNOS peptide completely eliminated eNOS-IR in the vasculature and in the eNOS-positive retinal neurons (B). At 22 WG, eNOS-immunostaining can only be eliminated by eNOS peptide (D), but not by nNOS peptide (C). An eNOS-IR vessel was located at the NFL (open arrowheads). (E) At 20 WG, eNOS expression was detected in intensively stained cell bodies in the central artery and in small vessels at the optic nerve head. This eNOS-staining was also eliminated by preabsorption with eNOS peptide. (A, C, insets) Higher magnification of boxed regions. Scale bar, 50 μm.

Figure 3.

 

Distribution of eNOS immunoreactivity in a 16-WG flatmount human retina. (A) eNOS expression in the vasculature was restricted to the vessels emerging at the superior and inferior poles of the optic disc (OD). (B) Higher magnification reveals eNOS-expressing cells in the area surrounding the incipient fovea of the temporal retina. Scale bar: (A) 500 μm; (B) 100 μm.

Figure 4.

 

A montage of a coronal section of human retina at 17 WG showing that eNOS (red) was not coexpressed in cells expressing ganglion cell marker neurofilament (green). eNOS was strongly expressed in the blood vessels in the optic nerve as well as the choroid (arrows). The eNOS-expressing cells are numbered to indicate their relative positions in the retina. There were 22 eNOS-expressing cells identified in the temporal retina compared with only 4 in the nasal retina. Insets: enlargements of the eNOS-positive cells in the GCL and proximal INL. These most likely represent displaced amacrine cells and amacrine cells, respectively, at the positions indicated by the numbers. At this developmental stage, eNOS expression in the vasculature was primarily restricted to the optic disc. eNOS-expressing retinal cells were dispersed throughout most of the retina. More eNOS-expressing cells were found in an area surrounding the incipient fovea (an area roughly defined by cell numbers 3–18). eNOS expression proceeded in temporal-to-nasal, middle (fovea)-to-central, and peripheral gradients during fetal retinal development. Scale bar, 1 mm.

Figure 5.

 

Expression of eNOS and CD34 in the 22-WG human retina. (A) CD34 (green), a marker of vascular endothelial cells, was strongly expressed in cells lining the blood vessels. (B) Expression of eNOS was shown by red immunofluorescent labeling. (C) In merged images, yellow represents colocalization of eNOS with CD34 (open arrowheads) and was evident in the blood vessels at the NFL-GCL boundary and in the choroid. eNOS-positive retinal cells (arrows) in the proximal INL and GCL did not coexpress CD34. Scale bar, 50 μm.

Figure 6.

 

Expression of eNOS and proliferating cell marker ki-67 in the 17 WG human retina. (A) Expression of ki-67 (green) was exclusively restricted to proliferating cells, mainly in the outer NBL. (B) eNOS-expressing cells (red, arrows) at the proximal NBL and GCL. (C) In merged images, the eNOS-positive cells (red) in the proximal NBL and GCL did not demonstrate any ki-67 expression (green). Some eNOS-labeled dotlike structures appeared in the NBL, probably representing nonspecific staining induced by the antigen retrieval procedure. Scale bar, 50 μm.

Figure 7.

 

eNOS expression in the developing rat retina. eNOS expression was detected only in the blood vessels, not in the retinal neurons. At (A) P0 and (B) at P7, eNOS expression was restricted to the blood vessels on the vitreous surface of the retina (open arrowheads) and in the choroid. (C) At P14 and (D) in adult, eNOS expression was detected in the secondary blood vessels (open arrowheads) of the proximal and distal INL, and in the radial vessels (C, open arrows), extending across different layers of the retina. Scale bar, 50 μm.

Figure 8.

 

Coexpression of eNOS with markers of specific types of retinal neurons in the 22-WG human retina. (A–D) Expression of eNOS was shown by red immunofluorescent labeling and expression of retinal cell-type–specific markers was marked with green immunolabeling. Colocalization of eNOS with a specific marker is indicated in yellow in merged images. eNOS expression in the blood vessels (red, open arrowheads) was located between the NFL and GCL and in the choroid. In addition, some eNOS-expressing cells were detected in the proximal INL and GCL (red, arrows). Insets: boxed regions from images in (A–D) at higher magnification. Left to right: cell-specific marker (green), eNOS expression (red), and merged images (yellow). (A) Coexpression of eNOS with NeuN, a general marker of neurons was evident in the GCL and proximal INL, indicating that these eNOS-expressing cells were neurons. Note that eNOS expression was localized in the cytoplasm, and NeuN-immunolabeling was in both the nuclei and the cytoplasm. (B) Coexpression of eNOS with β-tubulin III, a marker of RGCs, was not detected in the GCL, showing that eNOS was not expressed by RGCs. (C) Coexpression of eNOS with syntaxin 1A, a general marker of amacrine cells, was present in the GCL and proximal INL, demonstrating that eNOS was expressed by amacrine cells. (D) Coexpression of eNOS with GAD67, a marker of GABAergic neurons, in the proximal INL, indicates that eNOS-positive neurons were GABA-expressing subpopulation of amacrine cells. (A, D) eNOS expression was found in some photoreceptors in the ONL (red, arrowheads). Scale bars: 50 μm; insets: 10 μm.

Figure 9.

 

eNOS was expressed in cells expressing 7G6, a marker of primate cone photoreceptors, in the 28-WG human retina. (A, D) 7G6 expression (green) was primarily restricted to the ONL. (B, E) eNOS (red) was also expressed in the ONL. (C, F) eNOS was coexpressed with 7G6 in cells of the ONL. In merged images, the yellow color represents colocalization of eNOS with 7G6. eNOS and 7G6 coexpression occurred in some cone photoreceptor cell bodies (arrows, ONL) and outer segments (arrowheads, OS), but some eNOS single-positive outer segments were identified in the outer ONL (F, open arrowheads). Scale bar, 10 μm.

The authors thank Phillis Kau for assistance in the experiments, Johnny Leung for assistance with the figure preparation, and Tony Chan and Alla Li for assistance with the confocal microscopic imaging.