Identification of protein kinase C α- and tyrosine hydroxylase-immunoreactive cells in the microbat retina.

A growing number of studies have revealed the functional neuroarchitecture of the microbat retina and suggested that microbats can see using their eyes. To better understand the organization of the microbat retina, quantitative analysis of protein kinase C alpha (PKCα)- and tyrosine hydroxylase (TH)-immunoreactive (IR) cells was conducted on the greater horseshoe bat (Rhinolophus ferrumequinum) retina. As a result, PKCα immunoreactivity was observed in rod bipolar cells, consistent with previous studies on other mammalian retinas. PKCα-IR cell distribution in the inner nuclear layer showed regional differences in density, with the highest density found in the nasal retina. The average density of PKCα-IR cells was 10,487±441 cells/mm² (mean ± S.D.; n=4), with a total of 43,077±1,843 cells/retina. TH-IR cells in the Rhinolophus ferrumequinum retina could be classified into four types based on soma location and ramification in the inner plexiform layer: conventional amacrine, displaced amacrine, interplexiform, and intercalated cells. The majority of TH-IR cells were conventional amacrine cells. TH-IR cells were nonrandomly distributed at low density over the retina. The average density was 29.7±3.1 cells/mm² (mean ± S.D.; n=3), with a total of 124.0±11.3 cells/retina. TH-IR processes showed varicosities and formed ring-like structures encircling AII amacrine cells. Our study provides the foundation for understanding the neurochemical architecture of the microbat retina and supports the notion that the eyes do play a role in the visual system of microbats.

Characterization of retinal ganglion cell, horizontal cell, and amacrine cell types expressing the neurotrophic receptor tyrosine kinase Ret.

We report the retinal expression pattern of Ret, a receptor tyrosine kinase for the glial derived neurotrophic factor (GDNF) family ligands (GFLs), during development and in the adult mouse. Ret is initially expressed in retinal ganglion cells (RGCs), followed by horizontal cells (HCs) and amacrine cells (ACs), beginning with the early stages of postmitotic development. Ret expression persists in all three classes of neurons in the adult. Using RNA sequencing, immunostaining and random sparse recombination, we show that Ret is expressed in at least three distinct types of ACs, and ten types of RGCs. Using intersectional genetics, we describe the dendritic arbor morphologies of RGC types expressing Ret in combination with each of the three members of the POU4f/Brn3 family of transcription factors. Ret expression overlaps with Brn3a in 4 RGC types, with Brn3b in 5 RGC types, and with Brn3c in one RGC type, respectively. Ret+ RGCs project to the lateral geniculate nucleus (LGN), pretectal area (PTA) and superior colliculus (SC), and avoid the suprachiasmatic nucleus and accessory optic system. Brn3a+ Ret+ and Brn3c+ Ret+ RGCs project preferentially to contralateral retinorecipient areas, while Brn3b+ Ret+ RGCs shows minor ipsilateral projections to the olivary pretectal nucleus and the LGN. Our findings establish intersectional genetic approaches for the anatomic and developmental characterization of individual Ret+ RGC types. In addition, they provide necessary information for addressing the potential interplay between GDNF neurotrophic signaling and transcriptional regulation in RGC type specification.

Tyrosine hydroxylase positive perisomatic rings are formed around various amacrine cell types in the mammalian retina.

Dopaminergic neurons of the central nervous system are mainly found in nuclei of the midbrain and the hypothalamus that provide subcortical and cortical targets with a rich and divergent innervation. Disturbance of signaling through this system underlies a variety of deteriorating conditions such as Parkinson's disease and schizophrenia. Although retinal dopaminergic signaling is largely independent of the above circuitry, malfunction of the retinal dopaminergic system has been associated with anomalies in visual adaptation and a number of retinal disorders. Dopamine (DA) is released mainly in a paracrine manner by a population of tyrosine hydroxylase expressing (TH(+) ) amacrine cells (AC) of the mammalian retina; thus DA reaches virtually all retinal cell types by diffusion. Despite this paracrine release, however, the so called AII ACs have been considered as the main targets of DA signaling owing to a characteristic and robust ring-like TH(+) innervation to the soma/dendritic-stalk area of AII cells. This apparent selectivity of TH(+) innervation seems to contradict the divergent DAergic signaling scheme of other brain loci. In this study, however, we show evidence for intimate proximity between TH(+) rings and somata of neurochemically identified non-AII cells. We also show that this phenomenon is not species specific, as we observe it in popular mammalian animal models including the rabbit, the rat, and the mouse. Finally, our dataset suggests the existence of further, yet unidentified post-synaptic targets of TH(+) dendritic rings. Therefore, we hypothesize that TH(+) ring-like structures target the majority of ACs non-selectively and that such contacts are wide-spread among mammals. Therefore, this new view of inner retinal TH(+) innervation resembles the divergent DAergic innervation of other brain areas through the mesolimbic, mesocortical, and mesostriatal signaling streams. AII amacrine cells have been considered as the main targets of dopamine signaling in the mammalian retina owing to a characteristic ring-like innervation from dopaminergic (TH(+) ) amacrine cells (green) to somata of AII cells (red). In this study, we show the intimate proximity of TH(+) rings and somata of non-AII cells, including starburst-a amacrine cells (blue) and other unidentified amacrine cells (magenta). We find that this phenomenon is not species specific and it occurs in a number of popular mammalian animal models. We hypothesize that TH(+) ring-inputs target most amacrine cells non-selectively and thus it resembles the divergent dopaminergic innervation of other brain areas.

Neuroprotective role of superoxide dismutase 1 in retinal ganglion cells and inner nuclear layer cells against N-methyl-d-aspartate-induced cytotoxicity.

The N-methyl-d-aspartate (NMDA) receptor-induced apoptosis is implicated in the pathological mechanisms of neural tissues, increasing the release of reactive oxygen species (ROS), resulting in a type of apoptotic cell death called excitotoxicity. Although intrinsic mechanisms to remove ROS, such as antioxidant enzymes, are provided by the tissue, the association between NMDA-induced excitotoxicity and antioxidative enzymes is not well understood. In this study, we focused on superoxide dismutase 1 (SOD1), an antioxidant enzyme, and investigated the role of SOD1 in the NMDA-induced neuronal cell death in the retina. NMDA was intravitreally injected into wild-type (WT) and SOD1 total knock-out (SOD1-deficient) mice. The number of TUNEL-positive cells in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) counted in the retinal sections and flatmount retinas were significantly higher in the SOD1-deficient mice than the WT mice after NMDA injection. Visual function assessed by dark-adapted electroretinogram (ERG) showed that the amplitudes of a-wave, b-wave, and oscillatory potential 2 were significantly reduced in the NMDA-injected SOD1-deficient mice. The level of ROS in the GCL and INL, measured using dihydroethidium, and the number of positive cells for γ-H2AX, a marker for DNA double strand breaks, and 8-OHdG, a marker for DNA oxidation, in the GCL were significantly increased in the SOD1-deficient mice after NMDA injection. We also measured mRNA and protein levels of SOD1 and SOD2 in the retina of WT mice, to find that mRNA and protein levels of SOD1, but not SOD2, were significantly reduced after NMDA injection. SOD1 deficiency exacerbated NMDA-induced damage to the inner retinal neurons, and NMDA reduced SOD1 levels in the retina of WT mice. Therefore, SOD1 protected retinal neurons against NMDA-induced retinal neurotoxicity, and NMDA-induced SOD1 reduction may be involved in neuronal vulnerability to excitotoxicity.

Pasireotide (SOM230) protects the retina in animal models of ischemia induced retinopathies.

The neuropeptide somatostatin and selective analogs for the sst(2/5) receptor subtypes provided neuroprotection against retinal chemical ischemia ex vivo and AMPA [(RS)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide] induced retinal toxicity in vivo, when employed in micromolar concentrations (Mastrodimou et al., 2005; Kiagiadaki and Thermos, 2008). The aim of the present study was to investigate the neuroprotective properties of a new metabolically stable agent pasireotide (SOM230) in the above mentioned retinal models of ischemia. Adult Sprague Dawley (250-350 g) rats were employed. For the ex vivo experiments, retinal eye cups were incubated with PBS or the chemical ischemia mixture [iodoacetic acid (5 mM)/sodium cyanide (25 mM)] in the absence or presence of SOM230 (10(-7)-10(-5) M) alone or in the presence of the sst(2) antagonist CYN-154806 (10(-7) or 10(-5) M). In the in vivo model, the animals received intravitreally: PBS (50 mM), AMPA (42 nmol/eye) or AMPA (42 nmol) in combination with SOM230 (10(-7)-10(-5) M). Immunohistochemistry studies using antisera against bNOS, a marker for brain/neuronal NOS containing amacrine cells, protein kinase C (PKC) a marker for rod bipolar cells, and TUNEL studies in conjunction with FACS analysis were employed to examine retinal cell loss and protection. Chemical ischemia led to a loss of bNOS and PKC immunoreactivity which was reversed by SOM230. Partial and full protection of bNOS and PKC immunoreactive neurons, respectively, was observed even at the low concentration of 10(-7) M. The neuroprotective actions of SOM230 (10(-7) or 10(-5) M) were reversed by CYN-154806 (10(-7) or 10(-5) M, respectively). Similarly, SOM230 (10(-7), 10(-6), 10(-5) M) provided neuroprotection in the in vivo model. The dose of 10(-7) M prevented the loss of the bNOS cells and provided almost full protection. These data were substantiated by TUNEL staining and fluorescence-activated cell sorting (FACS) analysis. SOM230 appears very efficacious in its neuroprotective properties in both models of retinal ischemia affording neuroprotection at the concentration or dose of 100 nM. These data suggest that SOM230 might represent a useful pharmacological compound for the treatment of retinal disease.

Age-dependent morphology of NADPH diaphorase-positive amacrine cells in the mouse retina.

NADPH diaphorase-positive amacrine cells (NAC) were studied in retinal whole mount preparation of mice, ranging from 1 day to 30 months of age. Following a peak in number and size during early development at postnatal day 14, their number and distribution remained well preserved up to senescence. Functional considerations include immunological, vascular and neuro-modulating aspects.

Two types of tyrosine hydroxylase-immunoreactive neurons in the zebrafish retina.

The purpose of the present study is to identify the dopaminergic amacrine (DA) cells in the inner nuclear layer (INL) of zebrafish retina through immunocytochemistry and quantitative analysis. Two types of tyrosine hydroxylase-immunoreactive (TH-IR) cells appeared on the basis of dendritic morphology and stratification patterns in the inner plexiform layer (IPL). The first (DA1) was bistratified, with branching planes in both s1 and s5 of the IPL. The second (DA2) was diffuse, with dendritic processes branched throughout the IPL. DA1 and DA2 cells corresponded morphologically to A(on)(-s1/s5) and A(diffuse)(-1) (Connaughton et al., 2004). The average number of total TH-IR cells was 1088±79cells per retina (n=5), and the mean density was 250±27cells/mm(2). Their density was highest in the mid central region of ventrotemporal retina and lowest in the periphery of dorsonasal retina. Quantitatively, 45.71% of the TH-IR cells were DA1 cells, while 54.29% were DA2 cells. No TH-IR cells expressed calbindin D28K, calretinin or parvalbumin, markers for the various INL cells present in several animals. Therefore the TH-IR cells in zebrafish are limited to very specific subpopulations of the amacrine cells.

Transmembrane semaphorin signalling controls laminar stratification in the mammalian retina.

In the vertebrate retina, establishment of precise synaptic connections among distinct retinal neuron cell types is critical for processing visual information and for accurate visual perception. Retinal ganglion cells (RGCs), amacrine cells and bipolar cells establish stereotypic neurite arborization patterns to form functional neural circuits in the inner plexiform layer (IPL), a laminar region that is conventionally divided into five major parallel sublaminae. However, the molecular mechanisms governing distinct retinal subtype targeting to specific sublaminae within the IPL remain to be elucidated. Here we show that the transmembrane semaphorin Sema6A signals through its receptor PlexinA4 (PlexA4) to control lamina-specific neuronal stratification in the mouse retina. Expression analyses demonstrate that Sema6A and PlexA4 proteins are expressed in a complementary fashion in the developing retina: Sema6A in most ON sublaminae and PlexA4 in OFF sublaminae of the IPL. Mice with null mutations in PlexA4 or Sema6A exhibit severe defects in stereotypic lamina-specific neurite arborization of tyrosine hydroxylase (TH)-expressing dopaminergic amacrine cells, intrinsically photosensitive RGCs (ipRGCs) and calbindin-positive cells in the IPL. Sema6A and PlexA4 genetically interact in vivo for the regulation of dopaminergic amacrine cell laminar targeting. Therefore, neuronal targeting to subdivisions of the IPL in the mammalian retina is directed by repulsive transmembrane guidance cues present on neuronal processes.

Effect of intravitreal administration of somatostatin and sst2 analogs on AMPA-induced neurotoxicity in rat retina.

PURPOSE: The aim of the present study was to use an in vivo model of retinal excitotoxicity to investigate the neuroprotective effect of somatostatin (SRIF)-ergic agents. METHODS: Adult Sprague-Dawley rats (weight range, 250-300 g) intravitreally received (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA; 21, 42, 84 nmol/eye) or PBS (50 mM). Time-dependent responses were examined in animals that received AMPA (42 nmol/eye). Animals received AMPA (42 nmol) alone or in combination with SRIF (10(-5), 10(-4) M) or the sst-selective ligands lanreotide (sst(2),10(-5),10(-4) M), L-779976 (sst(2,)10(-6),10(-5), 10(-4) M), L-797591 (sst(1),10(-4) M), and L-803087 (sst(4),10(-4) M). Immunohistochemistry and TUNEL studies were used to examine retinal cell loss and protection. Immunochemistry, Western blot analysis, and radioimmunoassay assessed the viability of sst(2A) receptors and SRIF levels, respectively, in control and AMPA-treated tissue. RESULTS: AMPA (42 nmol) treatment resulted in total and major loss of ChAT and bNOS immunoreactivity, respectively, 24 hours after its administration. This loss was sustained up to 30 days for ChAT- and 8 days for bNOS-expressing amacrine cells. SRIF and the sst(2) receptors were not affected by AMPA. SRIF and the sst(2) analogs protected the retina from the AMPA insult in a dose-dependent manner, whereas activation of the sst(1) and sst(4) subtypes had no effect. TUNEL staining confirmed AMPA-induced retinal ischemia and L-779976 neuroprotection. CONCLUSIONS: These results demonstrate for the first time that SRIF and the sst(2) analogs, administered intravitreally, protect the retina from excitotoxicity. Further studies are essential to ascertain the therapeutic relevance of these results.

Expression of natriuretic peptide-activated guanylate cyclases by cholinergic and dopaminergic amacrine cells of the rat retina.

Recently, the natriuretic peptides were detected in the cholinergic and dopaminergic amacrine cells of the retina. We performed immunofluorescence labeling of rat retinal sections to examine the immunoreactivity of natriuretic peptide-activated guanylate cyclases (NPR-A and NPR-B) in the rat retina, in particular whether they were localized to dopaminergic and cholinergic amacrine cells. NPR-A and NPR-B immunoreactivity was detected in several layers of the retina including amacrine cells. In amacrine cells, both NPR-A and NPR-B were co-localized with tyrosine hydroxylase, a marker of dopaminergic cells. NPR-B, but not NPR-A, was localized to amacrine cells expressing choline acetyltransferase (ChAT), a marker of cholinergic cells. These findings suggest that natriuretic peptides have different regulatory systems in dopaminergic and cholinergic amacrine cells in rat retina.

Quantification and characterization of GABA-ergic amacrine cells in the retina of GAD67-GFP knock-in mice.

PURPOSE: Although the presence of gamma-aminobutyrate acid (GABA) in amacrine cells and its co-localization with other neuronal substances is well known, there exists only little information about their quantitative distribution in the mouse eye. The aim of the present study was to characterize GABA-ergic amacrine cells in the retina of the recently introduced glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mouse. METHODS: Whole mounts of the retina were prepared and the GFP-positive neurons quantified. Immunofluorescence staining was performed with antibodies against GABA, calbindin (CB), calretinin (CR), parvalbumin (PV), choline acetyl transferase (ChAT), tyrosine hydroxylase (TH), vesicular glutamate transporter (VGluT) 1, VGluT2 and VGluT3. RESULTS: Displaced GABA-ergic amacrine cells in the ganglion cell layer (GCL) showed a density of 1006 +/- 170 cells/mm(2). In the inner nuclear layer (INL), the density of amacrine cells was 8821 +/- 448 cells/mm(2) in the central region and 6825 +/- 408 cells/mm(2) in the peripheral region. GFP-positive amacrine cells co-localized with GABA (99%), CR (INL 18%, GCL 71.3%), CB (INL 6.3%), bNOS (INL 1%, GCL 4%), and ChAT (INL 17%, GCL 92.6%). No co-localization was seen with antibodies against PV, TH, and VGluT 1-3. CONCLUSIONS: This study presents the first quantitative data concerning the co-localization of GABA-ergic neurons in the mouse retina with various neuronal markers.

The sensitivity of light-evoked responses of retinal ganglion cells is decreased in nitric oxide synthase gene knockout mice.

We have shown previously that increasing the production of nitric oxide (NO) results in a dampening of visual responses of retinal ganglion cells (G. Y. Wang, L. C. Liets, & L. M. Chalupa, 2003). To gain further insights into the role of NO in retinal function, we made whole-cell patch clamp recordings from ganglion cells of neural type nitric oxide synthase (nNOS) gene knockout mice. Here we show that in the dark-adapted state, the sensitivity of retinal ganglion cell to light stimulation is decreased in nNOS knockout animals. The lowest light intensities required to evoke optimal responses and the average intensities that evoked half-maximal responses were significantly higher in nNOS knockouts than in normal mice. Retinal histology and other features of light-evoked responses of ganglion cells in nNOS mice appeared to be indistinguishable from those of normal mice. Collectively, these results, in conjunction with our previous work, provide evidence that increasing levels of NO dampen visual responses of ganglion cells, while a lack of nNOS decreases the sensitivity of these neurons to light. Thus, NO levels in the retina are capable of modulating the information that ganglion cells convey to the visual centers of the brain.

Endothelial nitric oxide synthase is expressed in amacrine cells of developing human retinas.

PURPOSE: To examine the expression and cellular distribution pattern of endothelial nitric oxide synthase (eNOS) in the developing human retina and to compare its expression with that in rats. METHODS: Expression of eNOS was examined by immunohistochemistry in retinas of humans ranging from 8.5 to 28 weeks of gestation (WG) and of rats. RESULTS: In the developing human retina, eNOS expression was first detected in the proximal margin of the neuroblastic layer in the incipient fovea-surrounding area at 12 WG. At 17 to 28 WG, eNOS-immunoreactive cells were located in the innermost part of the inner nuclear layer and in the ganglion cell layer, expanding to both temporal and nasal retinas and the processes projecting into the inner plexiform layer. These eNOS-positive cells coexpressed syntaxin and glutamate decarboxylase, and are probably GABAergic amacrine cells. The onset of eNOS expression in developing amacrine cells, however, preceded the invasion of retinal vasculature, long before vascular function involving these cells can be expected, suggesting that eNOS has a role not only in vasoregulation but also in retinal development. From 20 WG on, eNOS was also detected in the photoreceptors adjacent to the fovea. eNOS expression in amacrine cells and photoreceptors was observed in the central-to-peripheral and temporal-to-nasal gradients. However, in the developing rat retina, eNOS was expressed exclusively in the vascular endothelial cells. CONCLUSIONS: The results support that eNOS plays a role, not only in the regulation of vascular function but also in the process of retinal development in humans.

Up-regulated expression of neuronal nitric oxide synthase in experimental diabetic retina.

Nitric oxide (NO) can play either a neuroprotective or a neurotoxic role in diverse neurodegenerative conditions. This study investigated the differential expression of neuronal nitric oxide synthase (nNOS) in the streptozotocin-induced diabetic rat retina to clarify the involvement of NO produced from neurons in the early pathogenesis of diabetic retinopathy. A decrease in thickness of the outer retina was evident at 12 and 24 weeks after onset of diabetes. nNOS was immunolocalized in two subtypes of amacrine cells, displaced amacrine cells and in some bipolar cells in the normal retinas. The densities of each type of nNOS-expressing neuron showed no significant differences in the diabetic retinas with the exception of the bipolar cells. The numbers of nNOS bipolar cells at 12 weeks of diabetes increased threefold, showing dendritic polarity of nNOS expression. Protein levels of nNOS increased throughout the diabetic retinas reaching a peak value at 24 weeks of diabetes. Thus, diabetes up-regulates the expression of nNOS in bipolar cells, and NO from these cells may aggravate the degeneration of the outer retina in the diabetic retinas.

[Role of nitric oxide in apoptosis of retinal neurons in human fetuses].

The localization of NADPH-diaphorase (NADPH-d), inducible NO-synthase (iNOS) and TUNEL-immunoreactive neurons was studied in the retina of human fetuses in the I-III trimesters of pregnancy. High NADPH-d activity was found in internal segments of photosensory cells, amacrine and ganglion cells. The population of NADPH-d-positive amacrine cells included three types of neurons. Neurons of the 1st type had large size and scarce dendritic field, occupying the inner nuclear and outer plexiform layers. Small neurons of the 2nd type were located in the inner plexiform layer. Ectopic amacrine cells of 3rd type could be found in the outer part of the ganglion cell layer. High density of the NADPH-d-positive neurons was detected in the central portion of retina surrounding fovea centralis and the optic disk area. The activity of NADPH-d was found to grow progressively in ontogenesis and to correlate with the appearance of immunoreactive iNOS in neurons. Immunoreactive iNOS marked a subpopulation of amacrine and ganglion cells which appeared in weeks 20-21 of gestation and attained maximal immunoreactivity by the end of the III trimester. TUNEL-immunoreactive nuclei of the neurons with the signs of the apoptotic destruction were found in weeks 30-31 of gestation. The highest apoptotic index was found in the population of ganglion cells. The data obtained strongly suggest that NO is a factor, mediating the neuronal apoptosis during the critical period of a differentiation of interneuronal connections in the human retina.

Displaced GAD65 amacrine cells of the guinea pig retina are morphologically diverse.

The ganglion cell layer of mammalian retina contains numerous amacrine cells. Many belong to one type, the cholinergic starburst cell, but the other types have not been systematically identified. Using a new method to target sparsely represented cell types, we filled about 200 amacrine neurons in the ganglion cell layer of the guinea pig visual streak and identified 11 types. Ten of these resemble types identified in other species with somas in the inner nuclear layer, but one type has not been previously reported. Most of the types and nearly all the injected cells (95%) arborized low in the synaptic layer where they would co-stratify with various classes of ON ganglion cell. The displaced somas (7% of all amacrine cells) thus represent a heterogeneous pool, which are relatively accessible for study of their interactions with ON ganglion cells.

Contribution of nitric oxide-producing cells in normal and diabetic rat retina.

PURPOSE: To examine the immunohistochemical localization of L-arginine and L-citrulline and determine where and how nitric oxide (NO) is produced in the normal and streptozotocin (STZ)-induced diabetic rat retinas. METHODS: NO is produced when L-arginine is changed to L-citrulline by NO synthase (NOS). In normal and STZ-induced diabetic rats, using an immunohistochemical method, we examined the retinal distribution of L-arginine and L-citrulline after intracardiac perfusion. We studied the distribution of NOS after immersed fixation and analyzed the number of neuronal NOS (nNOS)-positive neurons. RESULTS: We observed L-arginine localization in the internal limiting membrane (ILM), the ganglion cell layer (GCL), and the inner nuclear layer (INL). L-Arginine immunoreactivity in the diabetic rat retinas was found in the inner plexiform layer (IPL), as well as in the normal retina. L-Citrulline immunoreactivity in the normal and diabetic retinas was observed in the ILM, the GCL, the IPL, and the INL. nNOS staining in the normal and diabetic rat retinas was observed in the GCL, the IPL and the INL. The number of nNOS-positive amacrine cells was less in the diabetic rat retinas. CONCLUSION: NO might be produced in the GCL and amacrine cells, which show immunoreactivity to L-arginine, L-citrulline, and nNOS. In the early stage of diabetic retinopathy in STZ rat retinas, diabetes disturbed the function of the nNOS-positive amacrine cells and reduced NO production via nNOS.

Advanced glycation end products induce death of retinal neurons via activation of nitric oxide synthase.

The purpose of this study was to determine whether advanced glycation end products (AGEs) are neurotoxic for cultured retinal neurons consisting mainly of amacrine cells, and to determine whether endogenous nitric oxide (NO) is involved in the toxicity. Cultured retinal neurons obtained from fetal Wistar rats (gestational age 19 days) were maintained in culture for 10 days, and then exposed to different concentrations of AGEs (0.02, 0.1, and 0.5 mg ml(-1)) in cultured media for different lengths of time. Both trypan blue exclusion and TUNEL assay were used to determine whether AGEs were neurotoxic, and NG-nitro-L-arginine methyl ester (L-NAME, 500 microM), a nitric oxide synthase (NOS) inhibitor, was used to determine whether NO was involved. Immunohistochemical analyses were performed to determine whether specific receptors of AGEs (RAGE) are present on cultured retinal neurons; caspase-3 was activated, and 3-nitrotyrosine was expressed on neurons treated with AGEs. Nitrite levels were measured in the supernatants of the media where neurons were incubated with AGEs. AGEs induced cell death in a time- and dose-dependent manner. TUNEL-positive cells and immunoreactivity to cleaved caspase-3 were enhanced on neurons following exposure to AGEs. L-NAME significantly suppressed the AGEs-induced neurotoxicity as assessed by both trypan blue exclusion and TUNEL assays. Activation of NOS was suggested by enhanced immunoreactivity to 3-nitrotyrosine on neurons and increased nitrite levels in the media incubated with AGEs. These results indicate that AGEs are neurotoxic to retinal neurons in culture through the activation of NOS. Apoptotic pathways may be in part involved in the death of the neurons.

Neuronal nitric oxide synthase (nNOS) positive retinal amacrine cells are altered in the DBA/2NNia mouse, a murine model for angle-closure glaucoma.

PURPOSE: To characterize retinal amacrine cell changes in eyes of DBA/2NNia (DBA) mice that develop an inherited angle-closure glaucoma. METHODS: DBA and non-glaucomatous C57BL/6J mice of different age groups (2 to 23 months of age) were studied and compared. Morphologic investigations included NADPH-diaphorase staining of retinal whole mounts and fluorescence immunohistochemistry of cryosections with antibodies against neuronal nitric oxide synthase (nNOS), tyrosin hydroxylase (TH), gamma aminobutyric acid (GABA), and vesicular acetylcholine transporter (VAChT). RESULTS: Immunohistochemistry of amacrine cell subpopulations in the retinae of DBA mice revealed no significant staining differences in the two mouse strains at all ages using antibodies against TH, GABA, and VAChT. However, staining with nNOS and NADPH diaphorase revealed significant differences between the DBA strain and the C57BL/6J mice. With the onset of elevated IOP and glaucoma beginning at around 6 months in the DBA mice, the total number of NOS positive amacrine cells continuously decreased from 1000 cells at 6 months of age down to 480 cells in animals older than 20 months of age, but did not decline in age-matched C57 mouse retinas. CONCLUSION: We previously described a parafoveal loss of nNOS positive amacrine cells in the monkey glaucoma model. The fact that there is also a significant decrease of nNOS amacrine cells in the glaucomatous mouse eye indicates a specific response of nNOS positive amacrine cells in glaucomatous retinopathy.

Voltage-gated K(+) channel subunits on cholinergic and dopaminergic amacrine cells.

The expression of voltage-gated K+ (Kv) channel subunits on rat retinal cholinergic and dopaminergic amacrine cells was studied using double immunofluorescence labeling and confocal laser scanning microscopy. Staining for Kv3.1b was found in the cholinergic cells, being present on the membrane of somata, and on the processes, but not in the dopaminergic cells. Kv4.3-immunoreactivity was localized on the somatodendritic compartment of the dopaminergic cells, but was not found in the cholinergic cells. Differential expression between the two cell types was not found for 10 other subunits tested. These results suggest that the Kv3.1b and Kv4.3 subunits may differentially contribute to the electrophysiological properties underlying distinct functions of these two retinal interneurons.