Nitric oxide synthase (NOS) catalyzes the formation of nitric oxide (NO) from l-arginine. In this study, the cellular localization of neuronal NOS (nNOS) activity in the human retina since fetal development was examined by immunohistochemistry. No detectable staining in the fetal retina was present at 14 weeks of gestation (wg), the earliest age group examined. A centro-peripheral gradient of development of nNOS immunoreactivity was evident at 16–17 wg, with the midperipheral retina showing nNOS immunoreactivity in most of the cell types and the inner plexiform layer while the peripheral part demonstrated moderate immunoreactivity only in the ganglion cell layer and photoreceptor precursors. A transient increase in nNOS immunoreactivity in the ganglion cells and Müller cell endfeet between 18–19 and 24–25 wg was observed at the time when programmed cell death in the ganglion cell layer, loss of optic nerve fibres as well as increase in glutamate immunoreactivity and parvalbumin (a calcium binding protein) immunoreactivity in the ganglion cells was reported. These observations indicate that programmed cell death of ganglion cells in the retina may be linked to glutamate toxicity and NO activity, as also suggested by others in the retina and cerebral cortex.

The presence of nNOS immunoreactivity in the photoreceptors from 16–17 weeks of fetal life to adulthood indicates other functions, besides their involvement in photoreceptor function of transduction and information processing.

1. Introduction

The intracellular messenger nitric oxide (NO) has been shown to modulate a number of cellular functions in both neuronal and non-neuronal cells as well as to act as a mediator of cell damage in ischaemic brain injury and a variety of neurological diseases (see reviews in Moncada 1992; Snyder 1992; Paakkari and Lindsberg 1995). Recent physiological studies have indicated the involvement of NO in impulse transduction in the outer retina and in the modulation of visual signal during retinal information processing. In the retinas of lower vertebrates, NO donors or inhibitors of neuronal nitric oxide synthase (nNOS) have been shown to modify light-induced membrane hyperpolarization in photoreceptors (Schmidt et al 1992; Kurreny et al 1994) and electrically-coupled horizontal cells (De Vries and Schwartz 1989; Miyachi
et al 1991) as well as to activate cGMP-gated conductances in on-bipolar cells (Scheills and Falk 1992). Recently, NO donors have been shown to modulate cGMP-gated conductances in ganglion cells isolated from rat retina (Ahmad et al 1994) as well.

NO is formed by the enzyme nitric oxide synthase (NOS) which catalytically transforms l-arginine into NO and citrulline (Palmer et al 1988). Immunohistochemical localization of NOS and histochemical demonstration of NADPH diaphorase (a co-factor of NOS) activity, have been used extensively as indicators of NO producing cells on the assumption that NO being a short-lived gaseous molecule, its biosynthetic enzyme and co-factor must be located either in the target cells or in cells adjacent to the target cells (Vincent 1994).

In the adult retina, NOS has been shown by labelling with antibody to its neuronal isoform – nNOS, to be localized in a small subset of amacrine cells and cells within the ganglion cell layer (Dawson et al 1991; Yamamoto et al 1993), rod photoreceptors (Venturini et al 1991), photoreceptor ellipsoids, the distal region of bipolar and glial cells apposing photoreceptor inner segments as well as in the inner and outer plexiform layers (Liepe et al 1994). A recent study by electron microscope immunocytochemistry has shown nNOS to be present in some bipolar and horizontal cell processes at the photoreceptor ribbon synapses in turtle and rat retina (Haverkamp and Eldred 1998). The development of the nNOS activity in retina has been studied in cat, rat and rabbit (Vaccaro et al 1991; Haberecht et al 1994; Perez et al 1995) with diverse observations. The present study has examined the developmental appearance and changes in the expression of NOS enzyme by immunohistochemical localization of constitutive nNOS in the retinas of human fetuses ranging in gestational ages from 14 to 25 weeks, a five-month-old infant and an adult subject (35 year-old).

2. Materials and methods

2.1 Tissue samples and fixation

Retinas were obtained from human fetuses of 14 weeks of gestation (wg, 2), 16–17 wg (3), 18–19 wg (1), 20–21 wg (3) and 24–25 wg (3) as well as from a five-month-old infant and a 35-year-old male subject. The fetuses were procured from cases of hysterotomy and legal spontaneous abortions, whereas the postnatal and adult samples were obtained from autopsies. The required clearance of the Institute’s ethical committee and consent of the concerned parents were taken for collection of the fetuses and their use (eyeballs) in research. The fetal ages were determined on the basis of the crown rump length (Hamilton et al 1972), the biparietal diameter and foot length. The retinas were immersion fixed in 4% paraformaldehyde or 2·5% glutaraldehyde for 3–4 days at 4°C. After washing in 0·1 M phosphate buffer saline (PBS), the tissues were cryoprotected in 15–30% sucrose overnight, embedded in tissue freezing medium (Leica) and cryosectioned at – 20^oC at 20–25 m m thickness.

2.2 Immunohistochemistry

The glutaraldehyde-fixed tissue sections were reduced with sodium borohydride (0·1%) for 10 min. The free-floating sections were incubated in 0·3% hydrogen peroxide (H₂O₂) in 90% methanol for 30 min to inactivate the endogenous peroxidase activity. After washing in 0·01 M PBS, sections were blocked for 4 h in 10% goat normal serum and then incubated in the primary antiserum for nNOS (1 : 500 dilution, rabbit polyclonal, Chemicon, Temecula, USA). The secondary antibody used was biotinylated goat anti-rabbit IgG (1 : 200 dilution, Vector Laboratories, Burlingame, CA, USA) for 6 h at 4°C. The antigen-antibody complex was localized employing an avidin-biotin-peroxidase system (ABC kit, Vector Laboratories, USA) according to the manufacturers instructions. Peroxidase staining was developed using 3,3˘ -diaminobenzidine tetrahydrochloride (0·06%) as chromogen, in 0·1 M acetate-imidazole buffer (pH 7·4) together with H₂O₂ (0·06%) and nickel sulphate (0·5%). To demonstrate the specificity of the antibody, negative control sections were incubated and processed similarly but with the omission of the primary antiserum. The retinas from two adult rhesus monkeys (Macaca mulatta), being available fresh from surgery, were also included in this study to better understand the cellular distribution of nNOS immunoreactivity in the primate retina as well serve as positive controls.

3. Results

The human retina follows a centro-peripheral gradient of development from close to the optic disc and shows differentiation of its various cell types in a vitreal to scleral direction. Hence the midperipheral and peripheral retinas from different gestational age periods, the five month-old infant and the adult subject (5–7 mm away from the fovea) were compared. Paraformaldehyde fixed-sections at 14 wg, the earliest age group examined in the present study, revealed negligible staining in the fetal retina (figure 1). At 16–17 wg, the midperipheral retina showed a number of nNOS-immunopositive cells in the ganglion cell layer, presumably ganglion cells and displaced amacrine cells. The vitreal endfeet of the Mü ller cells were clearly immunostained. Diffusely distributed reaction product was also seen in the processes of the Müller cells towards the outer limiting membrane as well as in their cell bodies lying near the incipient outer plexiform layer. The inner plexiform layer, and some of the amacrine cells located close to it were immunopositive. In addition, the photoreceptor precursor cells present in the outer neuroblastic zone near the ventricular surface showed faint immunostaining (figure 2A). In the peripheral part of retina, moderate immunoreactivity was observed in the majority of the neurons lying in the ganglion cell layer and photoreceptor precursor cells (figure 2B). At 18–19 wg, in the peripheral retina, while the intensity of immunoreactivity was enhanced in the ganglion cell layer and photoreceptor precursor cells, faint nNOS immunopositivity was detected in the cells of the innermost row of the outer neuroblastic zone – the prospective amacrine cells (figure 3A). The five-month-old infant retina showed nNOS immunoreactivity in the ganglion cell layer and in some scattered cells (presumably amacrine) of the inner nuclear layer. In the photoreceptor layer, the immunoreactivity was present in cell bodies and outer segments (figure 4A). In the adult human retina, the immunoreactivity was present strongly in the nerve fiber layer, and moderately in some amacrine cells and cone ellipsoids. Occasional cells in the ganglion cell layer were stained. In addition, both the inner and outer plexiform layers also showed faint nNOS immunoreactivity (figure 4B). In the monkey retinas fixed in paraformaldehyde, most cells of the ganglion cell layer, cone ellipsoids, inner fibres and the outer segments were strongly labelled (figure 5A, B), perhaps due to the better preservation of the antigen in the fresh retinas.

It is interesting to note that the paraformaldehyde preparations showed nNOS immunoreactivity in both the neuronal and glial elements; on the contrary, the glutaraldehyde-fixed retinal preparations from fetuses of 20–21 (figure 3B) and 24–25 wg (not shown) as well as from the adult human (not shown) and monkeys (figure 5C) showed prominent immunoreactivity mainly in the Müller glial processes and their end feet. Although some photoreceptor precursors were also stained in the early gestational age periods (see e.g., figure 3B), in the adult retinas, nNOS immunoreactivity was absent in the mature photoreceptors (figure 5C). The cell types labelled with nNOS at different ages and using different fixatives are comprehensively tabulated in table 1.

4. Discussion

4.1 Fixatives and nNOS

The present study has demonstrated that in retinas fixed in paraformaldehyde, nNOS immunoreactivity was revealed in both neurons and glia, while glutaraldehyde fixation preferentially allowed prominent staining of Müller glial cells though some neuronal elements (i.e., photoreceptor precursors) also were variably nNOS-immunoreactive. It is interesting to note that such an observation was also made by Huxlin (1995) when labelling adult rat retinal samples for NADPH-diaphorase activity with different fixatives and time protocols, suggesting thereby that the nature of fixatives, fixation conditions and exposure time are indeed critical factors in the cellular localization of NOS and its cofactor, NADPH-diaphorase. We have not stained sections for NADPH-diaphorase, but localization of NOS immunoreactivity is considered equivalent to the demonstration of localization of NADPH-diaphorase (Valtschanoff et al 1993).

4.2 Adult retina

The adult retina of different vertebrates consistently shows nNOS immunoreactivity and NADPH-diaphorase in the amacrine cells (Vaccaro et al 1991; Liepe et al 1994; Perez et al 1995). However, the localization of nNOS/NADPH-diaphorase in other cell types as well as in the outer and inner plexiform layers has not been consistently reported in different vertebrates and in the same animal species by different authors. In the present study, the adult human retina revealed staining in Müller glia, ganglion and amacrine cells, cone inner (ellipsoids) and outer segments, outer and inner plexiform layers, and nerve fibre layer. In the adult monkey, all components of the cone photoreceptors were more prominently stained in both the para- and peri-foveal region (figure 5A, B).

4.3 Developing retina

Studies on the developing rat retina have shown that although the mature expression of nNOS and its cofactor does not occur until the end of second postnatal week, the

developmental expression of the cellular label of NADPH-diaphorase or nNOS immunoreactivity begins at postnatal age three (Mitrofanis 1989) to seven days (Perez et al 1995). In rabbit and cat retinas, NADPH-diaphorase reactive cells, on the other hand, have been detected prenatally (Vaccaro et al 1991; Mitrofanis et al 1992). The activity of this second messenger system has also been shown to be present early in the chick retina (Ientile et al 1996) and in dissociated murine cortical cultures (Southam and Garthwaite 1991). The present study too, showed prenatal appearance of nNOS immunoreactivity in the human retina. At 16–17 wg, the nNOS antigen is exhibited by the ganglion and displaced amacrine cells as well as Müller glial endfeet in the inner retina and by the photoreceptor precursor cells at the ventricular surface.

4.4 Role for NO in developing and adult retina

A transient increase in nNOS-immunoreactivity in the ganglion cells and Müller cell endfeet of fetal human retinas between 18–19 and 24–25 wg has been observed in the present study at the time when naturally occurring programmed cell death is reported to occur in the developing human ganglion cell layer (Provis et al 1985a). The loss of ganglion cells in fetal human retina is substantiated by the concurrent excessive reduction in the number of optic nerve fibres (axons of ganglion cells) between 16–17 and 19–20 weeks of gestation followed by gradual loss up to 26 weeks of prenatal life (Provis et al 1985b; Wadhwa and Bijlani 1987). At 16–17 wg, glutamate immunoreactivity is also observed to increase in the retinal ganglion cell layer (Wadhwa et al 1994; Jotwani et al 1998) as well as a spurt is noted in parvalbumin (protein which binds glutamate released Ca⁺) immunoreactivity in fetal human retina (Nag and Wadhwa 1996). Collectively, these observations indicate that programmed cell death of ganglion cells which occurs in the retina may be linked to excessive glutamate and NO activity. Involvement of NO in cell death has been suggested in rat retina (Huxlin and Bennett 1995; Nichol et al 1995) and cerebral cortex (Bredt and Snyder 1994). In a recent study, Haberecht et al (1997) have examined the development of retinal susceptibility to glutamate toxicity as well as the protective effects of two N-methyl-d-aspartate (NMDA) antagonists and NOS inhibitor, N^G-methyl-l-arginine (met ARG) using one day in vitro retinal explants of adult and neonatal (postnatal day 1) rabbits. Their observations suggest that NMDA/NO excitotoxic pathway may not be active at birth since the NOS inhibitor had little effect at this stage, thereby indicating that NO may play a role other than that of a toxic agent in developing retina. In their earlier study, Haberecht et al (1994) have shown that NOS containing cells are present in their mature form only around second postnatal week. Thus it appears that since this study
was conducted in explants from postnatal day 1, the developmental neurotoxicity of glutamate/NO which peaks at the end of first postnatal week in rabbit may not have been addressed optimally by the authors. It is interesting to note that during the period of 16 to 26 wg in the human fetus, the next higher visual relay centre, i.e., lateral geniculate nucleus shows segregation of its cells and retinal terminals into the adult like laminar pattern (Damayanti et al 1983) which is dependent on correct matching and refinement of connectivity of optic nerve fibres coming from the retinal ganglion cells. There is evidence to show that the formation of activity-dependent synaptic connections and refinement of projections from retina to lateral geniculate nucleus in the ferret (Cramer et al 1996) as well as in developing and regenerating olfactory neurons (Roskams et al 1994) also occurs through glutamate via NMDA receptors and NO. A dose dependent role for NO similar to that shown for glutamate (Lipton and Kater 1989) resulting in dendritic growth and synapse development at low concentrations and its neurotoxic effects at high titres needs further evaluation.

Continued presence of NOS-immunoreactivity in the photoreceptors from 16–17 weeks of fetal life to adulthood indicates other functions besides their definitive involvement in the photoreceptor function of transduction and information processing.

Acknowledgements

This study was supported by research grant (SW) from the Council of Scientific and Industrial Research (CSIR), New Delhi. TCN received a Research Associateship from CSIR.

MS received 1 March 1999; accepted 15 September 1999