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

Shengxiu Li,1,2 David Tay,1 Siyun Shu,2 Xinmin Bao,2 Yongming Wu,2 Xiaoyang Wang,3 and Henry K. Yip1

PURPOSE. To examine the expression and cellular distribution molecules that are regulated by a series of processes including pattern of endothelial nitric oxide synthase (eNOS) in the synthesis, posttranslational modiﬁcation, formation of synaptic developing human retina and to compare its expression with vesicles, and regulation of release, the production of NO is that in rats. mainly regulated by a class of NOSs that catalyze the conver- METHODS. Expression of eNOS was examined by immunohisto- sion of L-arginine and oxygen to L-citrulline and NO. Of the chemistry in retinas of humans ranging from 8.5 to 28 weeks of three NOS isoforms—neuronal (n)NOS, endothelial (e)NOS, gestation (WG) and of rats. and inducible (i)NOS—eNOS originally occurs discretely in vascular endothelial cells and is mostly associated with the RESULTS. In the developing human retina, eNOS expression was potent vasodilator property of NO.6 ﬁrst detected in the proximal margin of the neuroblastic layer eNOS expression is not only found in vascular endothelial in the incipient fovea-surrounding area at 12 WG. At 17 to 28 cells, but also in neurons in selected regions of the central WG, eNOS-immunoreactive cells were located in the inner- nervous system (CNS).7–9 eNOS in the pyramidal cells of the most part of the inner nuclear layer and in the ganglion cell hippocampus has been shown to participate in the induction layer, expanding to both temporal and nasal retinas and the of long-term potentiation,9 a form of synaptic plasticity. eNOS processes projecting into the inner plexiform layer. These is expressed in neural stem and precursor cells and has been eNOS-positive cells coexpressed syntaxin and glutamate decar- implicated in the regulation of neuronal progenitor cell prolif- boxylase, and are probably GABAergic amacrine cells. The eration and differentiation.10 Astrocytes in the CNS respond to onset of eNOS expression in developing amacrine cells, how- viral infection by an increase in eNOS expression.11 These ever, preceded the invasion of retinal vasculature, long before observations have indicated that eNOS has important biologi- vascular function involving these cells can be expected, sug- cal functions in the nervous system, in addition to its role gesting that eNOS has a role not only in vasoregulation but also affecting vascular functions. in retinal development. From 20 WG on, eNOS was also de- As in other tissues, eNOS has been localized to endothelial tected in the photoreceptors adjacent to the fovea. eNOS cells lining the vasculature in the retina.12,13 In addition to the expression in amacrine cells and photoreceptors was observed presence of eNOS in the blood vessels, a recent study has in the central-to-peripheral and temporal-to-nasal gradients. demonstrated that cells in the ganglion cell layer (GCL) can be However, in the developing rat retina, eNOS was expressed induced to express eNOS after ischemia–reperfusion injury.12 exclusively in the vascular endothelial cells. Moreover, eNOS immunoreactivity (IR) was detected in retinal CONCLUSIONS. The results support that eNOS plays a role, not ganglion cells (RGCs) in the postnatal rat retina.14 Photorecep- only in the regulation of vascular function but also in the tors, amacrine cells, and RGCs in the developing chick retina process of retinal development in humans. (Invest Ophthalmol have also been shown to contain basal levels of eNOS.15 How- Vis Sci. 2006;47:2141–2149) DOI:10.1167/iovs.04-1202 ever, the expression of eNOS in the developing human retina is still unknown, and consequently, so is the role that eNOS n addition to its well-known role in regulating cardiovascular may have in the development of human retina. Therefore, the Ifunctions, nitric oxide (NO) has been implicated in a variety purpose of the present study was to examine the expression of physiological and pathophysiological processes in the ner- and cellular distribution pattern of eNOS in the developing vous system, such as neurotransmission, brain development, human retina, and thereby help to elucidate the possible role synaptic plasticity, trophic function, and glutamate-mediated of eNOS in retinal development. The expression pattern of toxicity.1–5 NO is a highly diffusible and short-lived signaling eNOS in the developing human retina is also compared with molecule that is produced on demand by activation of nitric that of the rat retina. oxide synthase (NOS). Unlike other intercellular messenger

METHODS From the 1Department of Anatomy, The University of Hong Kong, Pokfulam, Hong Kong SAR, China; and the Departments of 2Neurobi- Collection and Preparation of Human ology and 3Obstetrics and Gynecology, Zhujiang Hospital, Guangzhou, Fetal Tissues China. Supported by grants from the Committee on Research and Con- Ten human fetal eyes, ranging in age from 8.5 to 28 weeks of gestation ference Grant, the University of Hong Kong and the Regional Grant (WG), were obtained under approved protocols, and all study proce- Council of Hong Kong. dures were in accordance with the guidelines set forth in the Decla- Submitted for publication October 9, 2004; revised August 30 and ration of Helsinki and the terms of all relevant local legislation. The age November 8, 2005; accepted March 13, 2006. of the fetus was determined by a combination of medical records, Disclosure: S. Li, None; D. Tay, None; S. Shu, None; X. Bao, supersonic check, and crown–rump length. Fetuses older than 17 WG None; Y. Wu, None; X. Wang, None; H.K. Yip, None were perfused through the left ventricle with 0.9% saline, followed by The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS, pH ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. 7.4) at 4°C. The eyes were then quickly enucleated; cornea, lens, and Corresponding author: Henry K. Yip, Department of Anatomy, vitreous body were removed; and the eyecups were postfixed in the The University of Hong Kong, Pokfulam, Hong Kong SAR, China; same fixative for a further 24 to 48 hours. For fetuses at 12 to 17 WG, [email protected]. the eyecups were immersed in the same fixative for 1 to several days.

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For fetuses younger than 12 WG, whole eyes were fixed for several temperature. Control experiments included: primary antibody preab- days. The tissue was cryoprotected by immersion in 30% sucrose at 4°C sorbed with a 20-fold excess of a specific peptide mapping to the C- overnight, serially sectioned at 10 to 20 ␮m on a cryostat, mounted on terminal of human eNOS, which the eNOS antibody was raised against slides, and stored at Ϫ70°C. (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 Collection and Preparation of Rat Tissues company. 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 RESULTS in Teaching and Research and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Retinas from Expression of eNOS in Developing Human Sprague-Dawley rats were obtained at embryonic day (E)18, and post- Retinal Cells natal days (P)0, P7, and P14 and from adult (at least three animals at In the developing human retina, eNOS immunostaining was gen- each stage except only one animal at E18). Adult rats were anesthe- erally detected in endothelial cells of the blood vessels located on tized with 7% chloral hydrate (42 mg/100 g body weight, intraperito- neal 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-␮m-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 Biotech- nology, 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 ␤-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 Mo- lecular 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 (Vec- tastain Elite ABC kit; Vector Laboratories, Burlingame, CA) and developed Ј with 3,3 -diaminobenzidine (DAB) in 0.1 M acetic buffer (pH 6.5) con- FIGURE 1. Expression of eNOS during human retinal development. taining 1% ammonium nickel sulfate, 0.004% ammonium chloride, and The expression of eNOS in retinal sections was characterized by 0.0005% H2O2. For double-labeled immunofluorescent experiments, immunohistochemistry at different stages of prenatal retinal develop- eNOS antibody was detected with an amplification kit (Alexa Fluor 568- ment from 10 to 28 WG. (A) At 10 WG, eNOS expression was detected Tyramide Signal Amplification Kit; Invitrogen, Carlsbad, CA). The sections in the cells of the blood vessels on the vitreous surface of the retina (✱) were then incubated with antibodies for a second layer of labeling and and in the choroid only. (B) At 12 WG, in addition to the eNOS-stained then with Alexa Fluor 488–conjugated donkey anti-mouse IgG or anti- vessels, a few weak eNOS-staining cells are detected in the proximal arrows arrows rabbit IgG. The slides were mounted in antifade mounting medium (Dako NBL ( ). eNOS-positive cells ( ) were seen in the GCL and proximal margin of INL at 17 (C) and 28 (D) WG. Immunoreactive Corp., Carpinteria, CA) and observed under a confocal microscope (Bio- processes (arrowheads) can be seen projecting from these cells to- Rad Laboratories, Hercules, CA). Immunostaining of ki-67 was performed ward the IPL. (C) Secondary branch from the primary process of the as already described, except before incubation of primary antibody; sec- eNOS-IR cells in the proximal INL. (D) Note that a retinal vessel (open tions were heated in 0.01 M citrate buffer (pH 6.0) containing 0.3% Triton arrowhead) at the GCL also displayed eNOS-IR. (C, D, insets) Higher X-100 for 20 minutes at 85°C and then cooled for 30 minutes at room magnification of the boxed regions. Scale bar, 50 ␮m.

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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 ϳ1to 3at17WG. 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. Fur- thermore, 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 essen- tially confined to cells in the area surrounding the incipient

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)At20 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 ␮m.

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 18–21 FIGURE 3. Distribution of eNOS immunoreactivity in a 16-WG flat- studies ). From 17 WG onward, strong eNOS expression was mount human retina. (A) eNOS expression in the vasculature was found in cells located in the proximal inner nuclear layer (INL; restricted to the vessels emerging at the superior and inferior poles of probably corresponding to amacrine cells) and in the GCL (prob- the optic disc (OD). (B) Higher magnification reveals eNOS-expressing ably corresponding to ganglion cells or displaced amacrine cells; cells in the area surrounding the incipient fovea of the temporal retina. Figs. 1C, 1D). The eNOS-containing cells had round-to-ellipsoid Scale bar: (A) 500 ␮m; (B) 100 ␮m.

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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 rela- tive 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.

fovea (Fig. 3B). At this point in development, strong eNOS with CD34-IR. However, there was no CD34 coexpression in expression associated with the developing vasculature was the eNOS-labeled retinal cells in the GCL and INL (Fig. 5). To restricted in two vascular fronds emanating from the superior conﬁrm that the eNOS-expressing retinal cells are postmitotic and inferior poles of the optic disc (Fig. 3A). Thus, it is most neurons, we performed a double-labeling experiment with likely that eNOS is expressed by retinal cells in the intermedi- Ki-67, a cell marker for mitotically active cells (Fig. 6). We ate location of the incipient fovea and not by endothelial cells found that eNOS-positive retinal cells did not express Ki-67, associated with the developing retinal vasculature. indicating that eNOS was expressed in differentiating retinal More eNOS-positive cells were observed in the temporal cells and not in actively proliferating cells. These eNOS-IR half at 17 WG—mainly accumulating in the incipient fovea- retinal cells also displayed morphologic characteristics consis- surrounding area—than in the nasal half of the retina in a tent with the relatively mature neurons that have a round or horizontal section (Fig. 4). Thus, eNOS expression displayed a oval cell soma and nucleus and branching processes. temporal-to-nasal and a central-to-peripheral gradient. This spa- In the rat retina, at the age of E18 to adult, eNOS-immuno- tial expression pattern was in line with reports on the differ- labeled cells were detected only in the perivascular cells of entiation pattern of retinal neurons in the developing human blood vessels, and expression was absent in the retinal neu- retina (Li X, Yip HK, unpublished data, 2005, and a previous rons. At P0 and P7 (Figs. 7A, 7B), eNOS-IR vessels were found study21). At this stage, eNOS expression in the developing on the vitreous surface of the retina. At P14 and in adult eyes, vessels was still restricted to a small area surrounding the optic labeled vessels began to appear in deeper layers in the proxi- disc, to the optic nerve, and to the choroid. mal and distal INL and extended radially through the retina From 17 to 28 WG, even with the invasion of eNOS-stained (Figs. 7C, 7D). The labeling intensity of retinal vessels de- blood vessels, there was no close spatial relationship of the creased from P14 to adult, suggesting that eNOS expression eNOS-IR cells in the GCL and INL to the retinal vasculature. may be more related to development of vasculature in the rat Colocalization studies with CD34, the cell marker of vascular retina. All vessels in the choroid were also eNOS positive (Figs. endothelial cells, found that eNOS-labeled cell bodies, closely 7A–C). The results obtained in the present study on eNOS apposed to the retinal vasculature, overlapped extensively expression in the developing rat retina are in good agreement

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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 ␮m.

with those previously reported for the localization of eNOS-IR eNOS Expression in Photoreceptors in rat retina12 and also in accordance with previous findings on the development of retinal vessels in the rat.23 From 20 WG on, eNOS expression was also detected in some photoreceptors (Figs. 8A, 8D). Scattered eNOS-IR in the pho- Identity of eNOS-Immunoreactive Retinal Cells toreceptor layer was observed first in the incipient fovea- surrounding area and gradually extended into a larger area as To determine the identity of eNOS-IR cells, markers of specific development progressed. Some of the eNOS-positive photore- classes of retinal neurons were used in the double-labeled ceptors expressed 7G6, a marker for cone photoreceptors (Fig. immunofluorescence experiments with eNOS. We found that 9). Coexpression of eNOS and rhodopsin, a rod photoreceptor eNOS-positive cells in the GCL and INL adjoining the IPL were marker, was not confirmed, probably because of the limited colocalized with NeuN, indicating that these cells are neurons number of rhodopsin-positive rod photoreceptors detected at (Fig. 8A). However, they were not coexpressed with neurofila- this developmental stage and the poor integrity of the outer ␤ ment (Fig. 4) and -tubulin III (Fig. 8B), specific cell markers retinas in the specimens at 28 WG (data not shown). Thus, we for RGCs, suggesting that eNOS-stained cells in the GCL or INL cannot rule out the possibility that some of the eNOS-labeled were not RGCs. Coexpression of eNOS with syntaxin, a general photoreceptors are rod photoreceptors. 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 DISCUSSION and GCL, respectively. Furthermore, we sought to determine whether eNOS expression identifies amacrine cells expressing Two lines of evidence in recent studies have demonstrated the the glutamate decarboxylase (GAD) 67-kDa isoform (Fig. 8D). dynamic expression pattern of different NOS isoforms in the Extensive colocalization of eNOS with GAD67 occurred in this CNS. First, knockout mice carrying targeted mutations in the population of amacrine cells, indicating that eNOS-expressing nNOS genes display residual NOS in the brain.24 Second, eNOS, amacrine cells in the INL and GCL are GABAergic amacrine once thought to be present only in vascular endothelial cells, cells. has now been found to be expressed by neurons and glial cells

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 ␮m.

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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 subse- quent 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. Differ- FIGURE 7. eNOS expression in the developing rat retina. eNOS ex- ences in sensitivity between the antisera, differences in exper- pression was detected only in the blood vessels, not in the retinal imental conditions, or differences in species perhaps explains neurons. At (A) P0 and (B) at P7, eNOS expression was restricted to the the discrepancy. blood vessels on the vitreous surface of the retina (open arrowheads) In the present study, we used the most stringent controls in and in the choroid. (C) At P14 and (D) in adult, eNOS expression was immunohistochemistry to verify the specificity of the eNOS detected in the secondary blood vessels (open arrowheads)ofthe antibody, by performing the preabsorption with a peptide proximal and distal INL, and in the radial vessels (C, open arrows), specific to the eNOS antibody, or with a peptide specific to the extending across different layers of the retina. Scale bar, 50 ␮m. distinct sequence of nNOS, which is the closest related molecule to eNOS in structure and function. Preabsorption of the in the CNS.7–9 These results suggest that eNOS plays a role in eNOS antibody with eNOS peptides but not peptide specific to neural functions, in addition to the regulation of vascular ac- nNOS, completely abolished eNOS-IR. Furthermore, the eNOS tivities. In this study, we provide the first evidence showing antibody used in this study intensively labels blood vessels on that, in addition to vascular endothelial cells, eNOS is ex- the vitreous surface, in the retina, the choroid, and the optic pressed in a small population of retinal cells in the developing nerve head of both human and rat tissues. In addition, the human retina. Our immunohistochemical data demonstrate spatiotemporal pattern of eNOS expression in the developing that eNOS may have a dual role in both vascular and neuronal human retinal vasculature in our study conformed completely development of the human fetal retina. with observations in previous studies.25,26 The results from the Except for the demonstration of eNOS expression in retinal control experiments established the validity of our immunohis- neurons, our findings on the expression pattern of eNOS in the tochemical data. developing human retinal vasculature are in general agreement We demonstrated that, at 17 WG, eNOS-IR was primarily with previous observations.25,26 When eNOS-labeled amacrine localized in amacrine cells in the INL and displaced amacrine cells were first detected in the incipient fovea-surrounding area cells in the GCL and processes in the inner and outer plexiform at 12 WG, eNOS expression associated with retinal vasculature layer (OPL) at the incipient fovea-surrounding area and was restricted to the optic nerves. As development proceeded, eNOS-IR spread peripherally with increasing age (28 WG was when eNOS expression in amacrine cells had spread to the the latest gestation age examined). The appearance of eNOS- temporal and nasal retina, retinal vessels were still primarily expressing cells coincided with synaptogenesis in the IPL and localized to the area surrounding the optic disc, mainly at the the proportion of these cells increased concomitantly with superior and inferior retina. The fact that eNOS-expressing synaptic maturation, consistent with previous findings in hu- cells coexpressed NeuN but not CD34, a vascular endothelial mans. In the developing human retina, eNOS expression was cell marker, suggests that eNOS is expressed in retinal neurons observed in a temporal-to-nasal and central-to-peripheral gradi- and not in vascular endothelial cells during early fetal develop- ent, confirming the sequences of retinal maturation in many ment. The results revealed that the onset of eNOS expression species, including humans.21,29,30 eNOS-IR photoreceptors,

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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) Coex- pression of eNOS with ␤-tubulin III, a marker of RGCs, was not detected in the GCL, showing that eNOS was not expressed by RGCs. (C) Coexpres- sion 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) Co- expression 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 ␮m; insets:10␮m.

with immunolabeled cell bodies in the ONL and synaptic for- neuronal transmission from photoreceptors to ganglion cells in mation in the OPL, appeared in a similar manner and pro- the developing human retina. In this study, eNOS-labeled cells, ceeded in a temporonasal and central peripheral sequence. On apart from the endothelium of the blood vessels, can be found the whole, our results suggest that a wave of eNOS-positive only in human retina, not in rat retina. There may be species cells appears at the incipient fovea at ϳ17 WG and advances in variation in eNOS expression in the developing retina. a central–peripheral pattern across the retina as development Expression of NOS in the photoreceptors is still controver- progresses. Significantly, our results also suggest that this wave sial. Immunochemical and NADPH-diaphorase (NADPHd) his- of eNOS-expressing cells is coincident with a central–periph- tochemical staining failed to detect NOS in photoreceptors. eral pattern of synaptic formation in the IPL and OPL. This Other investigators have localized NOS in bovine photorecep- strongly suggests that eNOS expression in the INL, GCL, and tor inner segments. In contrast, NOS activity was said to be ONL is associated with the formation of synaptic contacts by present in the outer segments in another report. Our study amacrine cells, displaced amacrine cells, and photoreceptors in revealed the expression of eNOS in a small quantity of photo- the IPL and OPL, respectively. It has been shown that NO receptors expressing 7G6 in the ONL, suggesting that eNOS mediates the refinement of visual projections during develop- expression may identify early cone photoreceptors. NADPHd ment.31 In addition, NO appeared to participate in learning and reactivity has also been reported in a subpopulation of cone hippocampal synaptic plasticity.8,9 It is plausible that eNOS photoreceptors in adult human retina, possibly representing and NO in the retinal cells also function as retrograde messen- the blue cones. Furthermore, NADPHd histochemistry also gers in the refinement of synaptic connection and modulate identified the short-wavelength-sensitive (SWS or blue-sensi-

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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 infor- mation 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 sys- tems 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 sug- FIGURE 9. eNOS was expressed in cells expressing 7G6, a marker of gests that NOS-expressing amacrine cells participate in gluta- primate cone photoreceptors, in the 28-WG human retina. (A, D) 7G6 matergic circuits in the retina and that NOS may be triggered expression (green) was primarily restricted to the ONL. (B, E) eNOS by the activation of the NMDA receptor. The increase of intra- (red) was also expressed in the ONL. (C, F) eNOS was coexpressed cellular calcium, mediated by both L-type Ca2ϩ channels via with 7G6 in cells of the ONL. In merged images, the yellow color GABA receptors and by ligand-gated Ca2ϩ channels by NMDA represents colocalization of eNOS with 7G6. eNOS and 7G6 coexpres- receptors, is believed to be involved in a variety of develop- sion occurred in some cone photoreceptor cell bodies (arrows, ONL) 51 and outer segments (arrowheads, OS), but some eNOS single-positive mental events in CNS, such as neuronal migration and neu- 52,53 outer segments were identified in the outer ONL (F, open arrow- rite outgrowth. It is reasonable to speculate that mobiliza- ϩ heads). Scale bar, 10 ␮m. tion of intracellular Ca2 ions by both types of channels plays a role in activating eNOS in developing retinal cells and that the ϩ tive) cone in the cone-dominated retina of the tree shrew.32–36 spatiotemporal mobilization of the intracellular Ca2 mediated Thus, the eNOS-IR photoreceptors detected in this study may by different signaling pathways could influence eNOS activity contribute to the NOS activity detected with NADPHd histo- in a variety of developmental events in the retina. chemistry in the previous studies. These findings are consistent A significant finding in our studies was the apparent asso- with the morphologic characteristics and expression pattern ciation of eNOS expression with the development of retinal described for the SWS (S cones) in human fetal retina. Colo- neurons. Whether there is indeed a causal relationship be- calization experiments are needed to elucidate further the tween these two phenomena cannot be concluded from our identity of the eNOS-expressing cones.20 data. However, our data suggest that with the prenatal detec- This is the first report to describe the presence of eNOS in tion of eNOS expression in human fetal retina and with the subsets of neurons in the developing human retina. The spatial newly discovered role of neurotransmitters–modulators as and temporal sequence of eNOS expression correlates with the early signaling molecules for CNS development, the potential period of amacrine cells and photoreceptor differentiation and involvement of eNOS in neuronal development and differenti- synaptogenesis, consistent with a role for eNOS in retinal ation should be considered to be one of the possible functions development, in addition to the regulation of retinal circula- of the eNOS/NO system. tion. NO acts as a modulator of neuronal transmission in the mature nervous system, including the retina.37,38 Recent stud- Acknowledgments ies have suggested that in addition to their roles in synaptic communication in the mature brain, neurotransmitters and The authors thank Phillis Kau for assistance in the experiments, Johnny neuromodulators have a trophic role in neuronal maturation at Leung for assistance with the figure preparation, and Tony Chan and an early developmental stage. Thus, a neurotransmitter–neuro- Alla Li for assistance with the confocal microscopic imaging. modulator can take multiple forms and exert several actions at different developmental stages.39,40 Therefore, it is conceiv- References able that NO produced by retinal cells behaves in a similar 1. Bredt DS, Snyder SH. Transient nitric oxide synthase neurons in manner. It is, however, thus far unclear how NO participates in embryonic cerebral cortical plate, sensory ganglia, and olfactory neuronal development. 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