Immunocytochemical localisation of neuronal nitric oxide synthase in developing and transplanted rabbit retinas

 Nitric oxide (NO) acts as a modulator of neuronal transmission in mature neuronal systems, including the retina. Recently, NO has also been suggested to have a trophic function during development. We examined immunocytochemically the distribution of NO-producing cells in developing and transplanted rabbit retinas. An antibody detecting the neuronal isoform of its biosynthetic enzyme, nitric oxide synthase (NOS), was used on normal developing retinas [starting at embryonic day (E) 15] and on rabbit retinal transplants after various survival times (1–139 days after surgery). Weakly stained cell bodies were first observed in the proximal margin of the neuroblastic layer at E 29. Stained processes projecting towards a developing inner plexiform layer were also visible at this time point. Immunoreactive cells were located at later stages in the innermost part of the inner nuclear layer and in the ganglion cell layer, and are likely to correspond mainly to amacrine cells. NOS-labelled cells were also found in retinal transplants. The first NOS-labelled cells appeared, as in normal developing retinas, in ages corresponding to E 29 and were still detected in transplants corresponding to postnatal day 123. NOS-labelled cells were seen in areas between rosettes, where amacrine cells are located. NOS-labelled processes were at times seen to project for long distances, forming very distinct plexuses. NOS-containing amacrine cells thus appear both in the transplants and in developing retinas in the embryonic stages, long before synaptic function involving these cells can be expected, suggesting a role for NO not only in neuromodulation but also in retinal development. Accepted: 22 January 1997     

Retinal neuropeptides in the skates,Raja clavata,R. radiata,R. oscellata (Elasmobranchii)

Summary The distribution of immunoreactive neuropeptides was investigated in the retina of three species of skates (Raja clavata, R. radiata, R. oscellata), elasmobranch fish often used in electrophysiological work on the retina. Enkephalins, neuropeptide Y (NPY), substance P and glucagon were found in different types of amacrine cells.All four peptides appeared in cell bodies in the innermost part of the inner nuclear layer. Processes from the cells containing enkephalins were numerous and ramified throughout the inner plexiform layer. Processes from the cells containing glucagon were thick and rare, and were found throughout the inner plexiform layer, at times with a predominance in sublaminae 1 and 4. NPY-immunoreactive fibres appeared mainly in sublamina 1 but also in 2 or 3, and substance-P-immunoreactive fibres in sublaminae 1,4 and 5.Antisera against somatostatin, VIP or neurotensin did not show any immunoreactivity in the skate retina.     

Differential expression of syntaxin-1 and synaptophysin in the developing and adult human retina

Synaptophysin and syntaxin-1 are membrane proteins that associate with synaptic vesicles and presynaptic active zones at nerve endings, respectively. The former is known to be a good marker of synaptogenesis; this aspect, however, is not clear with syntaxin-1. In this study, the expression of both proteins was examined in the developing human retina and compared with their distribution in postnatal to adult retinas, by immunohistochemistry. In the inner plexiform layer, both were expressed simultaneously at 11–12 weeks of gestation, when synaptogenesis reportedly begins in the central retina. In the outer plexiform layer, however, the immunoreactivities were prominent by 16 weeks of gestation. Their expression in both plexiform layers followed a centre-to-periphery gradient. The immunoreactivities for both proteins were found in the immature photoreceptor, amacrine and ganglion cells; however, synaptophysin was differentially localized in bipolar cells and their axons, and syntaxin was present in some horizontal cells. In postnatal-to-adult retinas, synaptophysin immunoreactivity was prominent in photoreceptor terminals lying in the outer plexiform layer; on the contrary, syntaxin-1 was present in a thin immunoreactive band in this layer. In the inner plexiform layer, however, both were homogeneously distributed. Our study suggests that (i) syntaxin-1 appears in parallel with synapse formation; (ii) synaptogenesis in the human retina might follow a centre-to-periphery gradient; (iii) syntaxin-1 is likely to be absent from ribbon synapses of the outer plexiform layer, but may occur at presynaptic terminals of photoreceptor and horizontal cells, as is apparent from its localization in these cells, which is hitherto unreported for any vertebrate retina.     

Reorganization of horizontal cell processes in the developing FVB/N mouse retina

We investigated the morphological changes of horizontal cells after postnatal photoreceptor degeneration in the developing FVB/N mouse retina, using immunocytochemistry with anti-calbindin D-28K. From postnatal day 14 (P14) onwards, processes emerging from horizontal cells descend into the inner plexiform layer (IPL) and ramify mainly in stratum 1 of the IPL. Electron microscopy revealed that the descending processes make synaptic contacts with bipolar cells in the outer plexiform layer. Our results clearly demonstrate that loss of photoreceptor cells induces the reorganization of horizontal cell processes in the retinas of FVB/N mice as they mature.     

γ-Aminobutyric Acid System in Developing and Degenerating Mouse Retina

Freeze-dried sections (14 microns thick) of retinal layers were prepared from mice with retinal degeneration (C3H strain) and control mice (C57BL strain). The weighed sections (2-30 ng dry weight) were analyzed using our microassay methods. In the control retina, gamma-aminobutyric acid (GABA) concentration and glutamate decarboxylase (GAD) activity, on a dry weight basis, increased from birth to 9 weeks of age and decreased slightly at 20 weeks. In the degenerated retina, the levels of GABA and GAD activity were higher at birth than in the control retina, and continued to increase until 20 weeks of age, at which time the GAD activity reached a markedly high level. This increase was found when the total GABA and GAD levels per retina were determined. In the normal retinal layers, GABA and GAD were confined primarily to the inner plexiform layer. In the degenerated retina, GAD activity gradually increased in the inner layers during postnatal development, but by 20 weeks the increase was most prominent in the inner part of inner nuclear layer and in the outer part of inner plexiform layer. GABA transaminase activity and its distribution were not much different in both normal and degenerated retinas during development.     

Dopaminergic neurones in various retinas and the postnatal development of tyrosine-hydroxylase immunoreactivity in the rabbit retina

The localisation of tyrosine-hydroxylase immunoreactive neurones in retinas of a variety of animals were examined. Immunoreactivity was associated with specific populations of amacrine neurones in all species examined, viz; rabbit, guinea pig, monkey, cow, frog, pigeon and goldfish. Only in the goldfish was immunoreactivity also associated with processes situated in the outer plexiform layer showing that in this species catecholamine interplexiform cells exist. The development of tyrosine-hydroxylase immunoreactive neurones in the rabbit retina was also analysed. The first immunoreactive positive cells were observed by the third postnatal day. The immunoreactive positive neurones at this stage are weak and lack processes. The intensity of the immunoreactivity increases with development, but processes are lacking, until the 10th postnatal day. The immunoreactive neurones only appear fully developed by the 22nd to 28th postnatal day. Autoradiographical analysis of 3H-dopamine uptake strongly suggests that neurones containing tyrosine-hydroxylase immunoreactivity in the different retinas have the capacity to take up exogenous dopamine. It is therefore concluded that localisation of either 3H-dopamine uptake or tyrosine-hydroxylase provides a means of locating catecholamine neurones.     

Histological study of ibotenic acid-induced modifications of rat retina and their attenuation by diazepam

Summary After 2 h intraocular injections of 19 and 190 nmoles ibotenic acid in the rat retina produced an intensive vacuolization of the inner plexiform layer and cellular alterations, in the inner nuclear layer and ganglion cell layer. These alterations consisted of either cytoplasmic swelling accompanied by clumping of the nuclear chromatin or darkening of the cytoplasm along with nuclear condensation. A week later the retinas were thinner than the controls due to the disappearance of the affected cells. Pre-treatment with diazepam prevents the morphological alterations induced by 19 nmoles ibotenic acid; mainly the swelling, which was completely prevented, while the darkening was reduced drastically, although some vacuolization of the inner plexiform layer is still present.     

Neuropeptide Y (NPY)-like immunoreactive amacrine cells in retinas of frog and goldfish

Summary The distribution of neuropeptide Y (NPY)-like immunoreactivity in rat, rabbit, chick, frog and goldfish retinas was investigated by immunohistochemistry. Positive results were observed only in the frog and goldfish retinas. NPY immunoreactivity was associated with a small population of amacrine cell bodies in the inner nuclear layer and cell processes in the inner plexiform layer of both retinas. In the frog retina, three distinct layers containing immunoreactivity were observed in the inner plexiform layer. In contrast, the immunoreactivity in the same area of the goldfish retina was more or less separated into two layers. Convincing evidence could not be found for the co-existence of NPY-like material with other putative transmitter-like substances in the two retinas.Radioimmunoassay revealed the presence of small amounts of NPY-like immunoreactivity in the rabbit retina; the goldfish and frog retinas contained significantly more immunoreactive material. High performance liquid chromatography of the immunoreactive material in frog and goldfish retinas showed each retina containing different molecular forms of NPY-like proteins, neither of which resembled porcine NPY or PYY.The endogenous NPY-like material of the frog retina can be released by potassium depolarisation in a calciumdependent way. In view of all these data an NPY-like protein must now be considered a potential retinal transmitter.     

Distribution of calcitonin gene-related peptide immunoreactivity in the central nervous system of the forg,Rana esculenta

Summary Tyrosine hydroxylase (TH) immunocytochemistry was utilized to quantify dopaminergic synapses in the inner plexiform layer of the retina of Bufo marinus. Since dopaminergic cells have bistratified dendritic arborisation in the inner plexiform layer, attention was given to the segregation of synapses between the scleral and the vitreal sublaminae. Light-microscopically, a more elaborate dendritic branching was observed in the scleral than in the vitreal sublamina. In contrast, about 55% of synapses occurred in the vitreal one fifth of the inner plexiform layer, 30% in the scleral fifth, and 15% in the intermediate laminae. Input sources and output targets showed only minor quantitative differences between sublaminae 1 and 5. TH-immunoreactive processes were found in presynaptic (62.8%) and postsynaptic (37.2%) positions. Synapses to the stained dendrites derived from bipolar (40.4%) and amacrine (59.6%) cells, whereas outputs from the TH-positive processes were directed to amacrine cells (56.8%) and to small and medium-sized dendrites (35.4%); at least some of these can be considered as ganglion cell dendrites. TH-positive profiles neither formed synapses with each other nor were presynaptic to bipolar cell terminals. Junctional appositions of the immunoreactive profiles were occasionally seen on non-stained amacrine and ganglion cell dendrites in the scleral sublamina of the inner plexiform layer and on optic axons in the optic fibre layer. Although dopaminergic cells are mainly involved in amacrine-amacrine interactions, inputs from bipolar terminals and outputs to ganglion cell dendrites were also substantial, suggestive of a role also in vertical information processing.     

移植视网膜NOS阳性神经元的发育

目的 观察不同年龄组段大鼠正常视网膜及移植视网膜内NOS阳性神经元的发育情况及其定位分布。方法 实验分正常视网膜发育组和移植视网膜发育组,应用还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）组织化学方法显示。结果 1、NOS阳性神经元最早出现于生后第五天（P5）,P18时阳性神经元数目达到最高峰,2、移植视网膜具有正常视网膜的各层结构和相似的生长规律,NOS阳性神经元在生后第4天移植视网膜（TP4）中出现,TP12数量达到高峰值,TP22后降至正常成年鼠水平。结论 根据NOS阳性神经元的定位,分布,推测其为无长突细胞,移位无长突细胞及节细胞。     

The presence of dopamine-β-hydroxylase-like enzyme in the vertebrate retina

An analysis of the dopamine-β-hydroxylase activity in various ocular tissues revealed low enzymatic activity in all the retinas analysed. Bovine and monkey retinas had the highest enzyme activity. The enzyme is also present in bovine optic nerve and pigment epithelium/choroid complex, and low levels are present in the vitreous. Immunohistochemical analysis of various retinas with an antiserum raised to bovine adrenal dopamine-β-hydroxylase-showed positive staining only in monkey and bovine retinas. In both instances dopamine-β-hydroxylase immunoreactivity was associated with cells in the ganglion cell layer. These cells are probably ganglion cells since some positive staining was also observed in the bovine optic nerve. Autoradiographical analysis of the uptake of [³H]noradrenaline showed that grains were associated with some structures in the ganglion cell layer. A single prominent layer of terminals at the junction of the inner nuclear and inner plexiform layer of the bovine retina was also apparent. This was considered to be due to [³H]noradrenaline accumulation by dopaminergic amacrine cells. The results suggest that a small number of noradrenergic and/or adrenergic ganglion cells may exist in certain retinas.     

Dopaminergic neurones in various retinas and the postnatal development of tyrosine-hydroxylase immunoreactivity in the rabbit retina

Summary The localisation of tyrosine-hydroxylase immunoreactive neurones in retinas of a variety of animals were examined. Immunoreactivity was associated with specific populations of amacrine neurones in all species examined, viz; rabbit, guinea pig, monkey, cow, frog, pigeon and goldfish. Only in the goldfish was immunoreactivity also associated with processes situated in the outer plexiform layer showing that in this species catecholamine interplexiform cells exist.The development of tyrosine-hydroxylase immunoreactive neurones in the rabbit retina was also analysed. The first immunoreactive positive cells were observed by the third postnatal day. The immunoreactive positive neurones at this stage are weak and lack processes. The intensity of the immunoreactivity increases with development, but processes are lacking, until the 10th postnatal day. The immunoreactive neurones only appear fully developed by the 22nd to 28th postnatal day.Autoradiographical analysis of ³H-dopamine uptake strongly suggests that neurones containing tyrosine-hydroxylase immunoreactivity in the different retinas have the capacity to take up exogenous dopamine. It is therefore concluded that localisation of either ³H-dopamine uptake or tyrosine-hydroxylase provides a means of locating catecholamine neurones.     

Serotonin-containing neurones in vertebrate retinas

Abstract: It has been established by a combination of HPLC and electrochemical detection that frog, lizard, goldfish, rabbit, and bovine retinas contain both dopamine and serotonin. Immunohistological and immunoradiographical methods show that serotonin is localised in amacrine perikarya and processes situated in the inner plexiform layers of frog, lizard, and goldfish retinas. The amount of serotonin in the mammalian retina appears to be too low for detection in neurones. The serotonin in the bovine retina is located mainly in the inner nuclear and plexiform layers, suggesting that the amine is present in the same types of cells as found for frog, lizard, and goldfish retinas. Retinas incubated in [³H]serotonin showed that radioactivity is associated with processes in the inner plexiform layer and amacrine perikarya. These results suggest that the neuronal elements that contain endogenous serotonin also have the capacity to accumulate exogenous amine and are consistent with the opinion that serotonin has a neuronal function in retinas of a variety of vertebrates.     

Distribution of somatostatin receptor 5 in mouse and bullfrog retinas

Somatostatin (SRIF), as a neuroactive peptide in the CNS, may act as a neuromodulator through activation of five specific receptor subtypes (sst(1)-sst(5)). In this work we conducted a comparative study of the expression of sst(5) in mouse and bullfrog retinas by immunofluorescence double labeling. Basically, the expression profiles of sst(5) in the retinas of the two species were similar. That is, in the inner retina sst(5) was localized to dopaminergic and cholinergic amacrine cells, stained by tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) respectively, and cells in the ganglion cell layer, whereas in the outer retina immunostaining for sst(5) was observed in horizontal cells. However, a more widespread, abundant distribution of labeling for sst(5), as compared to mouse retina, was seen in bullfrog retina: strong labeling for sst(5) was diffusely distributed in both outer and inner plexiform layers (OPL and IPL) in the bullfrog retina, but the labeling was only observed in the IPL of the mouse retina. In addition, bullfrog photoreceptors, both rods and cones, but not mouse ones, were labeled by sst(5). In combination with the experiments showing that SRIF-immunoreactivity was mainly found in the inner retina, our results suggest that SRIF, released from SRIF-containing cells in the inner retina, may play a neuromodulatory role in both outer and inner retina mediated by volume transmission via sst(5) in bullfrog retina, while the SRIF action may be largely restricted to the mouse inner retina.     

GABA concentration and GAD activity levels in normal and degenerated retinas from mice

Freeze-dried sections (14 m thick) were prepared from mice with normal (C57BL strain) and degenerated (C3H strain) retinas. GABA concentration and GAD activity were determined in the microsamples (1.8–20 ng dry weight) of retinal layers and sublayers, using an enzymatic amplication reaction, NADP cycling. 1) GABA was distributed over all layers of normal retina with a broad concentration peak covering both inner nuclear and plexiform layers. In contrast, GAD activity was mostly localized in the inner plexiform layer. 2) GABA concentration was similar in one-fourth of the sublayers of each inner nuclear or plexiform layer. GAD activity was highest in the innermost sublayer of the inner nuclear layer. An increasing gradient of GAD activity was present in the inward direction in the inner plexiform layer. 3) In the degenerated retina, lacking in photoreceptors, the inner nuclear and plexiform layers remained, and GABA and GAD levels in these layers were similar to those in normal retina.Special Issue dedicated to Dr. O. H. Lowry.     

Targeting of amacrine cell neurites to appropriate synaptic laminae in the developing zebrafish retina

Cellular mechanisms underlying the precision by which neurons target their synaptic partners have largely been determined based on the study of projection neurons. By contrast, little is known about how interneurons establish their local connections in vivo. Here, we investigated how developing amacrine interneurons selectively innervate the appropriate region of the synaptic neuropil in the inner retina, the inner plexiform layer (IPL). Increases (ON) and decreases (OFF) in light intensity are processed by circuits that are structurally confined to separate ON and OFF synaptic sublaminae within the IPL. Using transgenic zebrafish in which the majority of amacrine cells express fluorescent protein, we determined that the earliest amacrine-derived neuritic plexus formed between two cell populations whose somata, at maturity, resided on opposite sides of this plexus. When we followed the behavior of individual amacrine cells over time, we discovered that they exhibited distinct patterns of structural dynamics at different stages of development. During cellular migration, amacrine cells exhibited an exuberant outgrowth of neurites that was undirected. Upon reaching the forming IPL, neurites extending towards the ganglion cell layer were relatively more stable. Importantly, when an arbor first formed, it preferentially ramified in either the inner or outer IPL corresponding to the future ON and OFF sublaminae, and maintained this stratification pattern. The specificity by which ON and OFF amacrine interneurons innervate their respective sublaminae in the IPL contrasts with that observed for projection neurons in the retina and elsewhere in the central nervous system.     

GABA release induced by aspartate-mediated activation of NMDA receptors is modulated by dopamine in a selective subpopulation of amacrine cells

Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the CNS, including the retina. In the chick retina, GABA is located in horizontal and amacrine cells and in some cells in the ganglion cell layer. It has been shown that glutamate and its agonists, NMDA, kainate, and aspartate, promote the release of GABA from isolated retina and from cultured retinal cells. Dopamine, the major catecholamine in the retina, inhibits the induction of GABA release by NMDA. Two to seven-day-old intact chicken retinas were stimulated with different glutamatergic agonists and the GABA remaining in the tissue was detected by immunohistochemical procedures. The exposure of retinas to 100 μ M NMDA for 30 minutes resulted in 50% reduction in the number of GABA-immunoreactive amacrine cells. Aspartate (100 μ M) treatment also resulted in 60% decrease in the number of GABA-immunoreactive amacrine cells. The number of GABA-immunoreactive horizontal cells was not affected by either NMDA or aspartate. In addition, dopamine reversed by 50% the reduction of the number of GABA-immunoreactive amacrine cells exposed to NMDA or aspartate. Kainate stimulation promoted a 50% reduction in the number of both GABA-immunoreactive amacrine and horizontal cells. Dopamine did not interfere with the kainate effect. While in control and in non-stimulated retinas a continuous and homogeneous immunolabeling was observed throughout the inner plexiform layer, retinas exposed to NMDA, kainate and aspartate displayed only a faint punctate labeling in the inner plexiform layer. It is concluded that, under our experimental conditions, both NMDA and aspartate induce the release of GABA exclusively from amacrine cells, and that the release is modulated by dopamine. On the other hand, kainate stimulates GABA release from both amacrine and horizontal cells with no interference of dopamine.     

Developmental expression of a synaptic vesicle-specific protein in the rat retina

In order to examine the appearance of synaptic vesicles and to correlate it with the formation of the synaptic layers, we have determined the staining pattern of a murine monoclonal antibody (SV 48) to a synaptic vesicle-associated protein in developing rat retina. The antigen was detected by the indirect immunofluorescence technique using cryostat sections of paraformaldehyde-fixed retinas. In the adult retina, the antibody stained both the outer plexiform (OPL) and the inner plexiform layers (IPL). The nuclear layers and the nerve fiber layer (NFL) were devoid of any staining. In prenatal and early postnatal (P) retinas, the antibody stained two bands which corresponded to the respective locations of the NFL and IPL. Staining in the NFL increased until P-4 and began to decline subsequently, and by P-8 little staining was left in this layer. In contrast, in the IPL, the intensity of staining increased gradually and leveled off by P-10. In the outer retina, a band of fluorescence corresponding to the OPL was first observed at P-5 and increased in intensity up to P-10. Immunoblotting studies showed that the major immunoreactive material from adult and embryonic retinas had a Mr approximately 65,000-67,000. As expected from its developmental pattern, all bands appeared initially in the central retina and subsequently in the peripheral retina. Our results show that the synaptic vesicle-protein is present in the nerve fiber layer before synaptogenesis in the central nervous system. Subsequently, the protein is lost from the NFL, possibly as a consequence of synapse formation.     

Autoradiographic identification of acetylcholine in the rabbit retina

Rabbit retinas were studied in vitro under conditions known to maintain their physiological function. Retinas incubated in the presence of [3H]choline synthesized substantial amounts of both [3H]phosphorylcholine and [3H]acetylcholine. With time, [3H]phosphorylcholine proceeded into phospholipids, primarily phosphatidylcholine. Retinas pulse-labeled by a 15-min exposure to 0.3 microM [3H]choline were incubated for a subsequent hour under chase conditions designed either to retain newly synthesized acetylcholine within synapses or to promote its release. At the end of this time the two groups of retinas were found to contain equal amounts of radioactivity in the phospholipid pathway, but only the retinas incubated under the acetylcholine-protecting conditions contained [3H]acetylcholine. Freeze-dried, vacuum-embedded tissue from each retina was autoradiographed on dry emulsion. All retinas showed silver grains over the photoreceptor cells and faint labeling of all ganglion cells. In the retinas that contained [3H]acetylcholine, silver grains also accumulated densely over a few cells with the position of amacrine cells, over a subset of the cells of the ganglion cell layer, and in two bands over the inner plexiform layer. Fixation of the retina with aqueous osmium tetroxide retained only the radioactive compounds located in the photoreceptor and ganglion cells. Sections from freeze-dried tissue lost their water-soluble choline metabolites when exposed to water, and autoradiography of such sections again revealed radioactivity primarily in the photoreceptor and ganglion cells. Radioactive compounds extracted from the sections were found to faithfully reflect those present in the tissue before processing; analysis of the compounds eluted from sections microdissected along the outer plexiform layer showed [3H]acetylcholine to have been synthesized only by cells of the inner retina. Taken together, these results indicate that the photoreceptor and ganglion cells are distinguished by a rapid synthesis of choline-containing phospholipids, while acetylcholine synthesis is restricted to a few cells at both margins of the inner plexiform layer. They imply that the only neurons to release acetylcholine within the rabbit retina are a small group of probable amacrine cells.