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Muller glia can act as endogenous stem cells and regenerate the missing neurons in the injured or degenerating retina in lower vertebrates. However, mammalian Muller glia, although can sometimes express stem cell markers and specific neuronal proteins in response to injury or degeneration, do not differentiate into functional neurons. We asked whether bFGF and insulin would stimulate the Muller glia to migrate, proliferate and differentiate into photoreceptors in rd1 mouse. We administered single or repeated (two or three) intravitreal injections of basic fibroblast growth factor (bFGF;200 μg) and insulin (2 μg) in 2-week-old rd1 mice. Muller glia were checked for proliferation, migration and differentiation using immunostaining. A single injection resulted within 5 days in a decrease in the numbers of Muller glia in the inner nuclear layer (INL) and a corresponding increase in the outer nuclear layer (ONL). The total number of Muller glia in the INL and ONL was unaltered, suggesting that they did not proliferate, but migrated from INL to ONL. However, maintaining the Muller cells in the ONL for two weeks or longer required repeated injections of bFGF and insulin. Interestingly, all Muller cells in the ONL expressed chx10, a stem cell marker. We did not find any immunolabeling for rhodopsin, m-opsin or s-opsin in the Muller glia in the ONL.

The neuroactive peptide enkephalin (ENK) has been postulated to play important roles in modulating visual information. The retinal presence of ENKergic cells has been revealed with conventional morphological protocols targeting ENK molecule especially in avian, however, the detailed distribution of ENKergic cells and their specific neurochemical features in the mammal retina remain unclear because of the difficulties in visualizing ENKergic cells efficiently and reliably. To address this question, we took advantage of the preproenkephalin-green fluorescent protein (PPE-GFP) transgenic mice previously generated and identified in our group, and identified the neurochemical characteristics of retinal ENKergic cells. The majority of ENKergic cells occupied the proximal inner nuclear layer with a few displaced in the ganglion cell layer. Further double labeling revealed that most of these ENKergic amacrine cells used inhibitory glycine or gamma-aminobutyric acid as the primary neurotransmitter. However, some of them also utilized excitatory glutamate as the primary neurotransmitter. The present findings suggest that the retinal ENKergic cells fall into a subpopulation of amacrine cells and show predominantly inhibitory as well as less dominantly excitatory neurochemical features. Our findings offered comprehensive morphological evidence for the function of ENKergic amacrine cells of mammal species.

We recently cloned the zebrafish neuronal enolase-2 gene and showed that a 12kb eno2 promoter element was sufficient to drive transgene expression widely in CNS neurons in vivo from 48 hours post-fertilization through adulthood. The aim of the present study was to establish the expression pattern of the 12kb eno2 promoter element in the zebrafish visual system. Endogenous eno2 mRNA was detected in the developing retina from 2 days post-fertilization (dpf), and by 12dpf was localized to the retinal ganglion cell, inner and outer nuclear layers. Similar to endogenous eno2, GFP expression in the retina of Tg(eno2:egfp)Pt404 larvae was first evident at 2dpf, and by 12dpf intense GFP expression was seen in the retinal ganglion cell and photoreceptor layers, with weaker expression in the inner nuclear layer. We identified cell types expressing the eno2 promoter element by using two complementary strategies: (i) double label immunofluorescence analysis of Tg(eno2:egfp)Pt404 zebrafish, and (ii) generation of double transgenic zebrafish expressing red fluorescent protein under transcriptional control of the 12kb eno2 promoter and GFP under a rod- or cone-specific promoter. The 12kb eno2 promoter was expressed in retinal ganglion cells, amacrine cells, including a subset that co-expressed tyrosine hydroxylase, and rod photoreceptors. These data suggest that abnormalities of vision should be sought in transgenic models of diseases generated using this promoter. Owing to the specific expression of fluorescent reporters in neuronal subpopulations, Tg(eno2:egfp)Pt404 and Tg(eno2:RFP) zebrafish may be useful for studies of retinal lamination, neuronal differentiation and synapse formation in the visual system.

The doublecortin (DCX) protein is associated with microtubules, and is essential for neuronal migration, differentiation, and plasticity. In mammals, it is expressed in developing neurons and new immature neuroblasts in the adult brain, but not generally in mature neurons. In the retina, doublecortin is detectable as early as embryonic day 15 (E15), is highly expressed between E18 and E20, and is poorly expressed postnatally. In this study, we investigated immunohistochemically the expression and cellular localization of doublecortin in the adult rat retina. Doublecortin was expressed in the outer plexiform layer (OPL), and in cells in the outer border of the inner nuclear layer (INL). No other layers were labeled by anti-doublecortin antibodies. In double-labeling experiments, doublecortin expression co-localized with the expression of the marker for horizontal cells, calbindin D. By contrast, the marker for immature neuroblasts, polysialylated neural cell-adhesion molecule, was not expressed in horizontal cells. These results suggest that either horizontal cells have the capacity to continuously remodel their neurites or doublecortin has a different function in horizontal cells from the control of neuronal plasticity that it is known to modulate other neurites. In addition, doublecortin might be an alternative molecular marker for horizontal cells in the adult rat retina.

Despite of their wide expression in the brain, the precise neurophysiological role of rat Eag1 (rEag1) and Eag2 (rEag2) K + channels remains elusive. Our previous studies in hippocampal pyramidal neurons demonstrate a somatodendritic localization of rEag1 and rEag2 channels, suggesting that the two channel isoforms may contribute to setting the membrane excitability of somas and dendrites. Here, we aim to further characterize the cellular and subcellular localization patterns of rEag1 and rEag2 proteins by studying their laminar distribution in the retina. Confocal microscopic analyses of immunofluorescence data revealed that rEag1 and rEag2 K + channels exhibit distinct cellular expression pattern in the retina. rEag1 immunoreactivity was most prominent in the outer half of the inner plexiform layer, whereas strong rEag2 immunostain was found in the outer and inner segments of photoreceptor cells, the outer plexiform layer, and the inner nuclear layer. These results suggest that rEag1 and rEag2 K + channels may play a significant role in the transmission of electrical signals along the retinal neuronal circuits. We also performed double-labeling experiments to demonstrate that rEag1 and rEag2 are predominantly expressed in the somatodendritic compartment of retinal neurons. In addition, we presented evidence suggesting that rEag1 channels may be expressed in the GABAergic amacrine cell. Finally, based on their different immunostaining patterns over the inner region of the retina, we propose that compared to rEag2, rEag1 expression encompasses a significantly broader range of the somatodendritic compartment of the retinal ganglion cell.

Elevated introcular pressure (IOP)-induced retinal neuron ischemic death includes an early phase of necrosis and prolonged phase of apoptosis. We used this ischemic model to observe the changes of sortilin and p75(NTR) protein expressions in rat retina. The results of Western blot analysis showed the expression of p75(NTR) at the band of 75 (mature form), 60 (non-glycosylated pieces) and 50 kDa (ectodomain shedding pieces), and the expression of sortilin at the 95 and 90 kDa (the mature form). The protein expressions of p75(NTR) (60 and 50 kDa pieces) and sortilin (90 kDa) increased significantly (p0.05) at days 3, 5 and 7 after retinal ischemia. This effect was also confirmed by immunofluorescence staining. Sortilin was primarily present in cell membrane of the ganglion cells layer (GCL) and large ganglion cell bodies by immunofluorescence labeling. There was little expression of p75(NTR) in the normal retina, while expression increased extensively in GCL, inner plexiform layer (IPL) and inner nuclear layer (INL) after retinal ischemia. p75(NTR) was shown to co-localize with neurofilament in the axons of neuronal cells by double-labeling. These results suggested that the protein expressions of 60 and 50 kDa forms of p75(NTR), and the 90 kDa mature form of sortilin increased in ischemia-induced retinal neuron of rats.

The mammalian Period1 gene is rhythmically expressed and its proteins are found within the nucleus of the cells of the suprachiasmatic nuclei (SCN), the central circadian pacemaker in mammals; however, whether the target of the PER1 proteins is also the nucleus in the retinal peripheral clock cells is yet to be determined. Using an anti-PER1 protein antibody in Western blot analyses, we found three isoforms (75, 110 and 140 kDa) in extracts of the SCN, as well as in other different parts of the brain, whereas just two isoforms (75 and 110 kDa) were detected in the retinal extracts. We have observed that PER1 immunolabelling has a cytoplasmic location in many cells of the ganglion cell layer and in a few cells in the inner nuclear layer of the mouse retina. This cellular location was seen in any of the tissue samples taken at 4 h intervals, either in the day/night cycle or in constant darkness, of both wild type and rd mice. Unlike this situation, PER1 isoforms were nuclear proteins in the SCN cells as well as in other parts of the brain of the same animals. No circadian changes were found for these clock proteins in the neural retina. These findings suggest that PER1 proteins play roles in the retina different from those established in the SCN.

Nicotinamide Adenine Dinucleotide Phosphate-Diaphorase (NADPH-d) expressing neurons in the retina of golden hamsters have been identified to be a subset of amacrine cells that provide a major source of Nitric Oxide (NO) in retina. This subset of amacrine cells in mouse retina was recently proved to contain the circadian clock gene Per1 (D.Q. Zhang, T. Zhou, G.X. Ruan, D.G. McMahon, Circadian rhythm of Period 1 clock gene expression in NOS amacrine cells of the mouse retina, Brain Res., 1050 (2005) 101-109). However, it remains unknown whether these clock-related NADPH-d amacrine cells can be regulated by light stimulation and thus synchronized to ambient day/night cycle. A previous study has reported that NADPH-d expressing amacrine cells in postnatal hamsters exhibited a surge after eye-opening (D. Tay, Y.C. Diao, Y.M. Xiao, K.F. So, Postnatal development of nicotinamide adenine dinucleotide phosphate-diaphorase-positive neurons in the retina of the golden hamster, J. Comp. Neurol., 446 (2002) 342-348) suggesting a possible effect of light on the NADPH-d amacrine cells. In order to further reveal the relationship between NADPH-d amacrine cells and light stimulation, the present study focuses on the changes of the expression of NADPH-d in the retina of postnatal hamsters reared in completely deprived light conditions. Prior to eye opening, P12 hamster pups were subjected to either bilateral eyelid suturing or dark rearing. On P28 a subgroup of light deprived hamsters was returned to lighting conditions and the expression of NADPH-d activities in the retina was assessed. In hamsters reared in the 12:12 light-dark cycle, the number of NADPH-d amacrine cells in the ganglion cell layer (GCL) increased right after eye-opening and reached the adult level gradually. However, hamsters subjected to both bilateral eyelid suturing and dark rearing, the number of NADPH-d amacrine cells in GCL was maintained at a low level but increased again upon returning to the 12:12 light-dark condition. In contrast, the number of NADPH-d expressing amacrine cells in the inner nuclear layer (INL) remained low and unaltered regardless of the lighting environment. This study demonstrates that there are two subpopulations of NADPH-d expressing amacrine cells with respect to different locations in the retina of hamsters. Different from those in INL, the NADPH-d amacrine cells in GCL of postnatal hamsters are dependent on the lighting environment implicating that these clock-related amacrine cells and the production of NO might be under a modulation of light stimulation.

In the developing chick retina, heat shock protein 108 (HSP108), which exhibits transferrin binding activity, has been demonstrated at the mRNA level, while transferrin shows two expression peaks. Here, we investigated the expression profile of HSP108 in the developing chick retina at the protein level. The localization of HSP108 in embryonic days 15 (E15), E18, and postnatal day 2 (P2) chick retina was examined immunohistochemically using monoclonal antibody 9G10 specific for chick HSP108, while the expression levels of HSP108 in developing chick retina from E12 to P2 and adult were measured by Western blot analysis. HSP108 was expressed in the ganglion cell layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, inner segments of photoreceptors and retinal pigment epithelium. Two peaks of HSP108 expression were found at around E13 and E18, respectively. Since the two HSP108 peaks appeared to be correlated with the transferrin expression peaks during retinal development, HSP108 may be associated with iron metabolism during the development of the retina.

In the retina, neurotransmission from photoreceptors to ON-cone and rod bipolar cells is sign reversing and mediated by the metabotropic glutamate receptor mGluR6, which converts the light-evoked hyperpolarization of the photoreceptors into depolarization of ON bipolar cells. The Royal College of Surgeons (RCS) rat retina undergoes progressive photoreceptor loss due to a genetic defect in the pigment epithelium cells. The consequences of photoreceptor loss and the concomitant loss of glutamatergic input to second-order retinal neurons on the expression of the metabotropic glutamate receptor was investigated in the RCS rat retina from early stages of photoreceptor degeneration (P17) up to several months after complete rod and cone degeneration (P120). The expression of the gene encoding mGluR6 was studied by in situ hybridization in the retina, using an [ 35 S]dATP-labeled oligonucleotide probe. In congenic control and RCS retina, we found mRNA expression of mGluR6 receptor only in the outer half of the inner nuclear layer (INL) on emulsion-coated retinal sections. Quantitative analysis of the hybridization signal obtained from the autoradiographic films revealed decreased expression levels of the mGluR6 mRNA at early stages of photoreceptor degeneration (P17). On the contrary, increased expression levels were observed at late stages of degeneration (P60 and P120) in RCS compared to congenic control retina. In conclusion, our data demonstrate that the metabotropic glutamate receptor-6 mRNA levels are altered in the young and adult RCS rat retina and suggest that the genetically induced degeneration of photoreceptors affects the expression of this receptor by the INL retinal neurons.

Natriuretic peptides (NPs) have been shown to modulate neuronal activities. By immunohistochemistry and confocal microscopy, we examined expression of atrial NP (ANP), brain NP (BNP) and C-type NP (CNP) in rat retina. Our results showed that these peptides were differentially expressed in the neural retina. While strong ANP-, BNP- and CNP-immunoreactivity (IR) was clearly seen in the outer and inner plexiform layers and on numerous neurons in the inner nuclear layer, BNP- and CNP-, but not ANP-IR, was present in some ganglion cells. Furthermore, ANP, BNP and CNP were expressed in Müller cells with distinct profiles, as shown by double labeling of NPs and vimentin. Labeling for BNP was rather strong in the main trunks, major processes, but hardly detectable in the endfeet. The expression profile for ANP was similar, but with a much lower level. On the contrary, the endfeet and major processes in the inner retina were strongly CNP-positive, with the main trunks and other major processes in the outer retina much less labeled. These results raise a possibility that NPs, when released from Müller cells, may perform layer dependent functions.

In a developmental study on the expression of heat shock protein 108 (HSP108) mRNA in the chicken retina, we found different spatial and temporal expressions of HSP108 mRNA in each retinal layer. While intense HSP108 signals were found in the retina neuroblast layer at embryonic day 5 (E5), the ganglion cell population (GC), inner nuclear layer (IN) and pigment epithelium (PE) showed HSP108 expression at E9. At E14, HSP108 signals were reduced versus the previous stages even though signals were still detected in the GC, the IN, the outer nuclear layer and the PE. HSP108 signals were still detectable at the E21 stage, although each retinal layer showed a much differentiated morphology and diminished signal intensity. These results suggest that HSP108 expression might be developmentally regulated throughout eye organogenesis and that it plays a role in ocular development.

In the present study the presence of choline acetyltransferase (ChAT) immunoreactive cells in the adult retina of Xenopus laevis was demonstrated and their appearance and maturation during development were examined. Two cholinergic amacrine cell types were identified in the retina. They were located in the deepest row of cells in the inner nuclear layer and in the ganglion cell layer, respectively. Cell processes from these cells organized distinct laminae within the inner plexiform layer. ChAT immunoreactivity was first observed at embryonic stage 35 coinciding with the onset of vision and increased rapidly in premetamorphosis as synaptogenesis and growth proceeded. The development of both ChAT cell populations occurred simultaneously and cells that expressed ChAT transiently were not observed. Our results contrast with previous studies that suggested a late involvement of acetylcholine in the retina of Xenopus and support the notion that, like in mammals, this transmitter is involved in early phases of neurogenesis, cell migration, neuronal growth, and synaptogenesis.

Betaxolol is a beta-adrenergic blocker but its neuroprotective action is generally thought to be due to its calcium channel blocking properties. In this study, we investigated neuronal cell damage and changes in the expression of neuronal nitric oxide synthase (nNOS) immunoreactivity in the ischemic retina and its relationship to the neuroprotection of betaxolol treatment after ischemic injury. Using the retina after ischemia, the expression of nNOS was studied by immunocytochemistry. In control retinas, two types of amacrine cells and a class of displaced amacrine cells were nNOS-labeled. After ischemia/reperfusion, the number of nNOS immunoreactive cells increased in both the ganglion cell layer and the inner nuclear layer compared to the control retinas. However, when experiments were carried out on animals that had been treated with betaxolol twice daily after ischemia/reperfusion, the number of nNOS immunoreactive cells decreased compared to the untreated ischemic retinas. These results suggest that an increase in nNOS expression could be associated with the degenerative changes in the ischemic retina, and that betaxolol treatment appears to play a role in protecting retinal tissue from ischemic damage.

The presence of thyrotropin-releasing hormone-immunoreactive (TRHir) amacrine cells is described for the first time in the retina of a teleost. These amacrine cells were mostly located in the inner nuclear layer, with occasional perikarya in the ganglion cell layer. Their processes formed a conspicuous plexus at the level of the ganglion cell perikarya. The TRHir amacrine cells appeared in posthatching stages, with the total number in retinas of juveniles approximately four times the number of cells in adults. Two types of TRHir cells, large and small, can be distinguished in developing stages, small cells outnumbering large cells. The TRHir cells of adults appears mainly to correspond to large, multistratified amacrine cells of developing stages. The possibility of transient expression of TRH in small amacrine cells during development is discussed.

The central neuroprotective effects against ischemic injury of fibroblast growth factor (FGF), administered either directly into the central nervous system or systemically, is well documented. Here we show in a rat model of transient retinal ischemia that the neuroprotective effect of systemically administered acidic fibroblast growth factor (aFGF, FGF-1) extends to the retina. Histological findings show a lower decrease of retinal ganglion cells and inner nuclear layer cells (P < 0.0001) in animals receiving FGF-1. These results suggest that FGF may function as a natural protection agent during transient retinal ischemia and further document that an efficient neuroprotection of central nervous tissues can be obtained by systemic administration of this protein. Our data may, thus, contribute to the development of novel and safe therapeutic approach for the treatment of the ischemic injury of the retina.

We examined cholinergic neurons in the developing rat retina; an antiserum against choline acetyltransferase (ChAT) and an antiserum against vesicular acetylcholine transporter (VAChT) were used. From postnatal day 4 (P4) to P10, ChAT- and VAChT-like immunoreactivities were seen in cells which were located in the outer part of the inner nuclear layer. These cells had relatively large cell bodies and extended several transversely oriented processes. Double fluorescence immunohistochemistry using an antiserum against calbindin D-28K, a specific marker for the horizontal cells, revealed that all of ChAT- or VAChT-labeled cells showed calbindin D-28K-like immunoreactivity. These cells were no longer immunostained after P11. Thus, acetylcholine was considered to be transiently synthesized in the horizontal cells during early postnatal developmental stages in the rat retina.

Lamprey retinal ganglion cells are localized in two separate layers: those close to the vitreous and those at the junction between the inner nuclear and inner plexiform layers, including some others in the inner nuclear layer, close to the photoreceptor cell layer. Whereas most ganglion cell dendrites arborize in the inner plexiform layer and contact amacrine, bipolar and retinopetal cell profiles, some of them, located in the inner nuclear layer, ascend radially through the outer plexiform layer and establish contacts with photoreceptor cells. This ganglion cell type might correspond to the biplexiform ganglion cells already described in gnathostome vertebrate species and could provide a fastforward signal from photoreceptors to ganglion cells, bypassing the usual bipolar cell interneuron.

Previous RT-PCR experiments revealed that the neural retina of the rat contains gene transcripts of numerous aquaporins (AQPs), including AQP6 (Tenckhoff et al., Neuroreport 16 (2005) 53-56). In the present study, we investigated the localization of AQP6 immunoreactivity in slices of the rat neural retina, and determined whether blue light injury of the retina affects the tissue distribution of this channel. AQP6 immunoreactivity was found to be selectively localized to the outer plexiform layer. Around the ribbon synapses in this layer, AQP6 labeling was co-localized with the glial water channel AQP4. AQP6 labeling was not colocalized with the marker of horizontal cells, calbindin, nor with the marker of rod bipolar cells, protein kinase Cα. Along with the degeneration of photoreceptor cells after blue light treatment of the retina, AQP6-labeled ribbon synapses disappeared, and a punctate AQP6 staining redistributed into the inner nuclear layer. The co-localization of AQP6 and the glial water channel AQP4 suggests a preferential localization of AQP6 in glial membranes that surround the ribbon synapses in the outer plexiform layer. AQP6 might be involved in the glia-mediated osmo and ion regulation of the extracellular space in this layer.

The expression of five genes (GluR A; B; C; D; GluR 5) encoding functional subunits of glutamate receptors was investigated in the rat retina using in situ hybridization with oligonucleotide probes. All five genes are expressed in the retina. All probes label cell bodies in the ganglion cell layer as well as somata in the inner third of the inner nuclear layer (INL), where the amacrine cells are located. In addition GluR 5, B and D, and to a lesser extent also GluR A are found in the middle and outer part of the INL, where bipolar and horizontal cells reside. Different subsets of retinal neurons may thus use glutamate receptors of different subunit composition.

[ 3 H]2-Deoxy- d -glucose-6-PO 4 ([ 3 H]2DG-6P) was visualised at the level of single cells in freeze-dried guinea pig retinal sections by in vitro light microscopic autoradiography after incubation with [ 3 H]2-deoxy- d -glucose ([ 3 H]2DG). In the dark, autoradiographs revealed heterogeneous labeling within individual retinal layers. Labeling, representing [ 3 H]2DG-6P, was preferentially located over Muller (glial) cells. Labelling over identified neurones in the inner nuclear layer, in contrast, was scarce and over ganglion cells was exceptional. Our observations indicate that Muller cells in the mammalian retina phosphorylate [ 3 H]2DG to [ 3 H]2DG-6P, the first step in glycolysis.

Tritiated thymidine ([3H]Tdr) was injected either intraocularly (i.o.) or intraperitoneally (i.p.). Autoradiography showed that more retinal cells were labeled by i.o. than by i.p. injection. Controls showed that the increased number of labeled retinal cells was not related to glioblast proliferation produced by trauma from the i.o. injection. After injection of identical doses, the peak concentration of [3H]Tdr available for incorporation into DNA was about 100 times greater after an i.o. than after an i.p. injection. The effective duration of the labeling pulse in goldfish was 9 times longer for an i.o. than an i.p. injection. This study shows that i.o. delivery of [3H]Tdr is preferable to i.p. injection for efficient delivery of label to proliferating retinal cells.

The ganglion cell layer of pre- and postnatal rat retina is positive for insulin immunoreactivity. At birth the inner nuclear layer also stains for insulin. By 5 days after birth the layers characteristic of the mature retina are demonstrable. At this time the outer nuclear layer and both limiting membranes show insulin reactivity. The lens is positive for insulin at all stages studied and the retinal pigment and choroid layers are positive after birth. These observations suggest that insulin may be important in differentiation and/or maturation of the retina.

The distribution of tyrosine hydroxylase immunoreactivity was investigated in retinae of metamorphic, postmetamorphic and adult lampreys. Immunoreactive cell bodies were located mainly in the innermost part of the inner nuclear layer, with a few cells scattered throughout the inner plexiform layer. The processes of these neurons ran preferentially in the inner plexiform layer. Additionally, dense plexus of labelled processes were observed in the outer plexiform and nuclear layers. These findings suggest that most of the tyrosine hydroxylase-immunoreactive cells in the lamprey retina are interplexiform cells.

In retinal ganglion cells (RGCs), the expression of various types of voltage-gated sodium channel (Nav) alpha-subunits (Nav1.1, Nav1.2, Nav1.3, and Nav1.6) has been reported. Like RGCs, certain subsets of retinal amacrine cells, including AII amacrine cells, generate tetrodotoxin (TTX)-sensitive action potentials in response to light; however, the Nav subtypes expressed in these cells have not been identified. We examined the Nav subtypes expressed in rat retinal amacrine cells by in situ hybridization (ISH) using RNA probes specific for TTX-sensitive Na(v)s (Nav1.1, Nav1.2, Nav1.3, Nav1.6, and Nav1.7). Our results confirmed that Nav1.1, Nav1.2, Nav1.3, and Nav1.6 are localized in the ganglion cell layer (GCL). Interestingly, Nav1.1 was expressed not only in the GCL, but also in the inner nuclear layer (INL). The cell bodies of the Nav1.1-positive cells in the INL were located at the INL/inner plexiform layer (IPL) border. The cell bodies of AII amacrine cells are located close to the INL/IPL border, and these cells can be labeled with antibodies against parvalbumin (PV). Therefore, we combined ISH with immunohistochemistry and discovered that most of the PV-immunoreactive cells located at the INL/IPL border express Nav1.1. Our results show that AII amacrine cells express Nav1.1.

Oxidative stress affects numerous intracellular macromolecules, and may result in cell death unless precisely regulated. Unregulated oxidative stress can be controlled by various cellular defense mechanisms such as glutathione (GSH) which can critically counteract the damaging effects of oxidative stress in mammalian cells. We determined the effects of unregulated oxidative stress induced by GSH depletion on cells in mouse retina. Mice were intraperitoneally injected with buthionine sulphoximine (BSO) at 1.5 g/kg. After 0, 1, 4, and 7 days of BSO administration, retinas were excised and sections were subjected to GSH assay and terminal uridine deoxynucleotidyl nick end labeling (TUNEL) analysis. After 4 days of BSO administration, the number of TUNEL positive cells was significantly increased. However, after 7 days, TUNEL positive cells returned to the basal level. The retinal region most affected by the BSO treatment appeared to be the outer nuclear layer where the photoreceptor cells reside. Different from cells in other regions, retinal cells in the inner nuclear layer increased in their apoptosis even after the first day of BSO injection, and the increase was further potentiated after 4 days. Taken together, our studies suggested that GSH depletion may cause unregulated oxidative stress to the cells in the retina and indeed increased cell death in the retina. The cells in the inner nuclear layer seemed to be affected earlier than the cells in other layers of the retina. The GSH level in the retina may be a crucial therapeutic target in preventing blindness.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide with a wide range of effects in the central and peripheral nervous systems. PACAP has well-documented neurotrophic and neuroprotective actions in both in vitro and in vivo models of different neuronal injuries. The aim of the present study was to investigate the possible neuroprotective effect of PACAP in retinal degeneration induced by monosodium-glutamate (MSG) in neonatal rats. Preceding the MSG treatment, PACAP (1 or 100 pmol/5 μl) was injected unilaterally into the vitreous body on postnatal days 1, 5 and 9. Immediately after the PACAP treatment, pups were treated with 2 mg/g body weight MSG subcutaneously. At 3 weeks of age, rats were sacrificed and retinas were removed and processed for histological examination. Our results show that MSG treatment caused severe degeneration, primarily of the inner retinal layers. The thickness of the entire retina was only approximately half of that of the normal retinas, and the inner nuclear layer seemed to be fused with the ganglionic cell layer, with no discernible inner plexiform layer. Retinas of animals treated with 1 pmol PACAP showed a similar degree of degeneration. However, retinas of rats treated with 100 pmol PACAP showed significantly less damage, with clearly distinguishable inner retinal layers. In summary, our present study shows that local PACAP treatment could attenuate the retinal degeneration induced by the excitotoxic effects of glutamate.

Expression patterns of mRNAs of L-AP4-sensitive metabotropic glutamate receptors (mGluR4, mGluR6, mGluR7) in the rat retina were examined by northern blot analysis and in situ hybridization histochemistry. Expression patterns of mGluR4 and mGluR7 mRNAs were quite different from that of mGluR6 mRNA which was expressed at the outer part of the inner nuclear layer. The mGluR4 mRNA was expressed on the cell bodies of the ganglion cells, but not in the inner or outer nuclear layer. The expression of mGluR7 mRNA was observed throughout the entire region of the inner nuclear layer and on the cell bodies of the ganglion cells.

Carassius RFamide (C-RFa) is a novel peptide, isolated originally from the brain of the Japanese crucian carp and sharing homologies with mammalian prolactin-releasing peptide (PrRP). It has been demonstrated previously that C-RFa mRNA is abundant in the proximal half (fundus) of the Japanese crucian carp eye. In the present work, we localized C-RFa by immunohistochemistry mainly to perikarya, in the proximal half of the inner nuclear layer (amacrine cell layer). This distribution is different from that of FMRFamide, which is confined to axon terminals of terminal nerve efferent fibers in the inner plexiform layer. Electrophysiological recording revealed that C-RFa depolarized some amacrine cells and hyperpolarized L-type horizontal cells in the carp. These results suggest that C-RFa is produced within the cyprinid retina and functions as a transmitter or neuromodulator in retinal image processing.

Using in situ hybridization, we investigated the expression of ciliary neurotrophic factor receptor ((CNTFRalpha) mRNA in the rat retina rendered ischemic by elevation of the intraocular pressure (IOP). The IOP was increased to 120 mmHg and maintained for 60 min. The rats were sacrificed on the day of reperfusion (DRP) 1, 3, 7, 14, and 28. In the normal retina, the signal for CNTFRalpha mRNA was present in retinal cells in the inner nuclear layer (INL) and in the ganglion cell layer (GCL). On DRP 1, numerous cells in the INL and GCL showed a CNTFRalpha mRNA signal. From DRP 3 onwards, CNTFRalpha mRNA appeared in photoreceptor cells located in the outer part of the outer nuclear layer. The signal in these cells increased up to DRP 14 and then decreased at DRP 28. Our findings suggest that cells expressing CNTFRalpha mRNA may resist the degenerative processes induced by ischemic insult in the rat retina.

We performed a comparative study on the distribution of neuronal nitric oxide synthase (nNOS) immunoreactivity and mRNA in a normal rat retina using immunocytochemistry and in situ hybridization technique. As in previous studies, we found NOS immunoreactive (NOS-IR) cells and fibers in inner and outer plexiform layers (IPL and OPL), inner nuclear layer (INL) and inner photorecetptor segment (IPS). However, very little nNOS-IR could be detected in groups of amacrine cells and ganglion cells localized in ganglion cell layer (GCL). However, in situ hybridization showed that intense NOS mRNA signals were mainly found in the GCL and INL while weak or no mRNA signals were detected in IPL, OPL, outer nuclear layer (ONL) and IPS. This difference suggests that nNOS proteins may be transported through axons into the terminals in the IPL and OPL after they were synthesized with nNOS mRNA templates in the INL. In the case of nNOS mRNA in GCL, synthesizing nNOS proteins may move outside the eyeball and carry out tasks in central nervous system. The difference of nNOS mRNA and nNOS IR means that the complete concurrence of nNOS IR and in situ hybridization results may not always occur in the rat retina.

In order to determine the cellular localization of GABA A α and β subunits in the hamster retina, single and double immunocytochemistry was performed in perfuse-fixed hamster retina using commercially-available antibodies against the two receptor subunits and glutamic acid decarboxylase. Strong GABA A β immunoreactivity was found in two strata of the inner plexiform layer and in perikarya of amacrine cells and bipolar cells in the inner nuclear layer. In contrast, no GABA A α immunoreactivity was detected. All but a few of the GABA A β -immunoreactive amacrine cells were found not to display glutamic acid decarboxylase immunoreactivity. The present results indicate that there is a differential localization of GABA A α and β subunits in different neuronal subpopulations in the hamster retina.

A detailed study about the distribution of nitric oxide synthase (NOS) isoforms, neuronal NOS (nNOS) and macrophagic NOS (mNOS), in normal rat retina was performed using immunocytochemistry by employing specific antibodies. The nNOS immunocytochemistry showed immunoreactive amacrine cells, fibres in inner and outer plexiform layers (IPL and OPL) and an immunostained band corresponding to inner photoreceptor segments (IPS). This was in agreement with NADPH-d histochemical results. mNOS immunoreactivity was found in cell somas localized in both, inner nuclear layer (INL) and ganglion cell layer (GCL), in slender Muller cell processes along IPL and GCL and also in the band corresponding to IPS. A different distribution of nNOS and mNOS was found in rat retina although both isoforms of NOS are co-localized in IPS.

An anti-Fos protein antiserum was used to elucidate the diurnal expression of Fos protein in the normal and degenerate rd/rd mice retina. We have found that Fos expression is stimulated in cells of both inner nuclear layer (INL) and ganglion cell layer (GCL) at the onset of light period and reaches its maximum after 2 h. After which, the number of stained nuclei decreases along the light/dark cycle until almost no reaction is observed at the end of dark period. This expression pattern was similar in both normal and rd/rd mice although degenerate retinas showed a much lower number of stained nuclei. Aged rd animals also show Fos expression in GCL and INL in response to light stimuli suggesting that severely degenerate retinas are still able to transduce light stimulus.

Although Thy-1 is one of the most abundant surface glycoproteins in mammalian central neurons, its functional role is still not clarified. In this study we demonstrate that Thy-1 expression in the rat retina starts only at embryonic day 19 on both the cell somas and processes of retinal ganglion cells. Thy-1 immunoreactivity in the ganglion cell layer increases until postnatal day 14 and is paralleled by an increase in the thickness of the inner plexiform layer. Thus, the beginning of Thy-1 expression is correlated with a developmental stage when axonal elongation and neurite sprouting of ganglion cells has finished and refinement of synaptic structures in the colliculus superior begins. From postnatal day 9 on, Thy-1 immunoreactivity can also be observed on cell somas at the border of the inner plexiform/inner nuclear layer.

We have amplified partial complementary DNAs for the chicken gamma-aminobutyric acid type C (GABAC) receptor rho 1 and rho 2 subunits using the polymerase chain reaction. These nucleotide sequences have been utilized to design specific oligonucleotide probes for the in situ hybridization localization of the corresponding messenger RNAs (mRNAs) in the 1-day-old chick retina. Although both transcripts are found almost exclusively in the inner nuclear layer, their distributions differ markedly. From the locations of the hybridization signals, we deduce that the rho 1-subunit mRNA is present mainly in bipolar cells and that the rho 2-subunit mRNA is present in both amacrine and horizontal cells. These results suggest that the rho 1 and rho 2 subunits frequently occur in different receptor complexes and, therefore, that subtypes of the GABAC receptor exist.

The localization of trkB and low-affinity nerve growth factor receptor (LNGFR) mRNAs in the developing rat retina was examined by in situ hybridization. TrkB mRNA was expressed in the ganglion cell layer (GCL), in the inner border of the neuroblastic layer (NBL), and the inner border of the inner nuclear layer (INL). LNGFR mRNA was expressed in the GCL, in almost full thickness of the NBL, and in the intermediate part of the INL. Although both trkB mRNA and LNGFR mRNA were expressed in the GCL, the expression pattern was different between these mRNAs; trkB mRNA was expressed in almost all cells in the GCL uniformly and the expression of LNGFR mRNA varied greatly from cell to cell. In addition, the expression of both mRNAs, especially LNGFR mRNA seemed to be down-regulated at P7, when programmed cell death of the RGCs was prominent. These observations indicate that LNGFR may modulate the function of trkB and that trkB and LNGFR play important roles in the development and maintenance of the RGCs.

The expression of mRNA coding for brain-derived neurotrophic factor (BDNF) and for its functional receptor, the full-length tyrosine kinase receptor trkB (trkB mRNA), was examined in early postnatal and adult rat retina by in situ hybridization using digoxygenin and radioactively-labeled oligonucleotide probes. BDNF and trkB mRNAs are expressed in the ganglion cell layer at postnatal-days (PN) 1, 4, 7, 14, 60, in proximal neuroblastic layer (PN 1, 4, 7), and proximal inner nuclear layer (PN 14, 60). Subpopulations of developing and mature retinal cells are thus capable of synthesizing BDNF.

The chicken retina contains a population of prominent elements in the inner nuclear layer, which stain for NADPH-diaphorase. In distribution and morphology, these elements resemble the terminals of the centrifugal projection from the isthmo-optic nucleus. This identification was confirmed by showing that the NADPH-diaphorase-positive elements in the retina degenerated after destruction of the isthmo-optic nucleus or tract. These results indicate that the centrifugal projection to the retina in birds uses nitric oxide as a messenger or transmitter, in addition to a more conventional but as yet unidentified transmitter.

We found cells expressing c-kit receptor and its ligand (Sl factor, SLF) in the retina of rats and mice. c-kit messenger RNA (mRNA) was detected in a limited number of cells in the inner nuclear layer of rats and mice by in situ hybridization. The c-kit-expressing cells were assumed to be a subpopulation of the amacrine cells on the basis of their size and distribution pattern. c-kit protein-containing cells were demonstrated in the mouse retina by using ACK2 monoclonal antibody against the extracellular domain of the mouse c-kit receptor. The c-kit protein was detected in cell bodies and processes of the presumed amacrine cells. Hybridization signals for SLF mRNA were observed in the retinal ganglion cells. The results suggest that the c-kit receptor and its ligand may play some roles in the formation of junctions between the amacrine and retinal ganglion cells.

The isthmo-optic nucleus (ION) is the major origin of the centrifugal fibers projecting to the avian retina. The retinopetal fibers from the ION were labeled with biocytin and examined light- and electron-microscopically in the Japanese quail. The terminals were seen on cell bodies in the inner part of the inner nuclear layer. These neurons innervated with the isthmo-optic fibers appear to constitute a separate population different from the ‘ordinary’ amacrine cells.

The appearance of calbindin D-28K, a calcium-binding protein, during development of the trout retina was studied by immunohistochemistry. The first calbindin immunoreactive cells appear in the inner nuclear layer at the equator of the embryonic retina at the stage 227 degrees C (around embryonic day 15). Just before hatching, stage 440 degrees C (around embryonic day 30) cells located in the ganglion cell layer and inner nuclear layer, expressed calbindin. This pattern of immunoreactivity was conserved in post-embryonic retinae (alevins 15 days old). In adult retinae the ganglion cells showed a faint immunoreaction; the amacrine cells are markedly fewer and their immunoreaction declined; and the bipolar cells expressed calbindin for the first time. The results obtained in the present work attending to the expression of calbindin, generally conforms with the vitreal to scleral progression of differentiation of the teleost retina. Ganglion, amacrine and bipolar cells undergo further maturation after beginning calbindin expression.

The cellular localization of a dopamine- and cAMP-regulated phosphoprotein of M r 32,000 (DARPP-32) was investigated in cat, monkey and human retina by immunohistochemistry. In cat, DARPP-32-immunoreactive cell bodies identified as Muller cells were demonstrated in the inner nuclear layer (INL) with processes closely surrounding the cell some of photoreceptors in the outer nuclear layer. Some DARPP-32-IR cells were also seen in the nerve fiber layer (NFL) sending processes to the inner plexiform layer. In monkey and human retina, DARPP-32-IR cell bodies were also demonstrated in the INL, with few cells located in the NFL.

Acidic fibroblast growth factor (aFGF) mRNA has been detected in adult mouse or bovine retina by in situ hybridization with bovine aFGF cDNA clones. It is localized on ganglion cell layer, inner nuclear layer, photoreceptors and slightly on pigmented epithelium. This synthesis of aFGF in highly specialized retinal cell types is discussed in the framework on current views about the role of FGF in retinal cell biology.

Alternate serial 1 micron Durcupan resin sections of tiger salamander retina were stained with antisera against GABA and glycine using postembed immunocytochemical techniques. Although the vast majority of neurons were labeled by either GABA or glycine antiserum, a small percentage of presumed amacrine cells in the inner nuclear layer and cells in the ganglion cell layer were clearly labeled by both antisera, indicative of colocalization of endogenous GABA and glycine. Although there is a greater than 90% chance that a labeled cell will be clearly labeled for either GABA or glycine immunoreactivity, the possibility for cotransmission of two inhibitory transmitters must be considered for a small percentage of these retinal neurons.

The ultrastructure of ganglion cells and centrifugal fibers of the larval lamprey retinas were studied using horseradish peroxidase (HRP) as a marker. Larval ganglion cells were found both in the inner nuclear layer and the inner plexiform layer of the differentiated retina, and also were present in the undifferentiated retina. Direct photoreceptor-ganglion cell contacts and the presence of centrifugal fibers are described for the first time in the lamprey. The centrifugal fibers contact directly with ganglion cells in this species.

The number and distribution of putative cholinergic amacrine cells in cat retina was demonstrated by immunocytochemical localization of choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. In whole mount preparations of the cat retina ChAT-immunoreactive neurons were found in the inner nuclear layer (presumably amacrine cells) and in the ganglion cell layer (presumably displaced amacrine cells). The density of ChAT-labelled neurons increased from about 280 cells/mm2 in peripheral retina, to about 2750 cells/mm2 in the central area. There were always more ChAT-positive cells in the ganglion cell layer (50-70%) than in the amacrine cell layer. Two narrow dendritic strata were labelled in the inner plexiform layer.

We have used Lucifer Yellow for histological tracing of displaced amacrine cells within the ganglion cell layer (GCL) during the embryonic development of the chicken retina. Incubating whole eyes in the dye leads to bright staining of all displaced amacrine cells, whereas ganglion cells and glial cells are not stained. A subpopulation of cells of the inner part of the inner nuclear layer (INL) are also stained (for further details see ref. 13). Kainic acid, which is known to interfere with and kill amacrine cell systems, blocks the staining of these cells fully. This in addition to histological evidence confirms that the LY-stained cells in the GCL are displaced amacrine cells. Of the cells in the GCL, 23% (+/- 3%) are of the displaced amacrine type. Further, we find that the cytoarchitectural arrangement of these cells changes significantly during development.

This study has examined the development of cells in the rat retina which contain nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase. NADPH-diaphorase cells were first detected at postnatal day (P) 3, in somata located in the inner part of the cytoblast layer (CBL). At this age, NADPH-diaphorase reactivity was also seen in weakly labelled fibers in the presumptive outer plexiform layer (OPL). By P5, the somata of most labelled cells were in the inner part of the inner nuclear layer (INL), and by P11, their processes had spread extensively within the inner plexiform layer (IPL). By P25, there was a striking change in the pattern of NADPH-diaphorase reactivity. First, cells had lost reactivity from their large and extensive dendrites and second, there was a distinct reduction in the diameters of labelled somata. Thus, NADPH-diaphorase reactivity was most prominent during the period of synaptogenesis in the IPL. Labelled cells at P3 numbered 120 and were largely found at the superior margin of the retina. By P11, their total number had increased to the adult value of about 3400 and their density was highest in peripheral retina. With further development, the differential expansion of the retina appeared to lower the peripheral densities, resulting in an approximately uniform distribution by adulthood.