Muscarinic signaling influences the patterning and phenotype of cholinergic amacrine cells in the developing chick retina.

BACKGROUND: Many studies in the vertebrate retina have characterized the differentiation of amacrine cells as a homogenous class of neurons, but little is known about the genes and factors that regulate the development of distinct types of amacrine cells. Accordingly, the purpose of this study was to characterize the development of the cholinergic amacrine cells and identify factors that influence their development. Cholinergic amacrine cells in the embryonic chick retina were identified by using antibodies to choline acetyltransferase (ChAT). RESULTS: We found that as ChAT-immunoreactive cells differentiate they expressed the homeodomain transcription factors Pax6 and Islet1, and the cell-cycle inhibitor p27kip1. As differentiation proceeds, type-II cholinergic cells, displaced to the ganglion cell layer, transiently expressed high levels of cellular retinoic acid binding protein (CRABP) and neurofilament, while type-I cells in the inner nuclear layer did not. Although there is a 1:1 ratio of type-I to type-II cells in vivo, in dissociated cell cultures the type-I cells (ChAT-positive and CRABP-negative) out-numbered the type-II cells (ChAT and CRABP-positive cells) by 2:1. The relative abundance of type-I to type-II cells was not influenced by Sonic Hedgehog (Shh), but was affected by compounds that act at muscarinic acetylcholine receptors. In addition, the abundance and mosaic patterning of type-II cholinergic amacrine cells is disrupted by interfering with muscarinic signaling. CONCLUSION: We conclude that: (1) during development type-I and type-II cholinergic amacrine cells are not homotypic, (2) the phenotypic differences between these subtypes of cells is controlled by the local microenvironment, and (3) appropriate levels of muscarinic signaling between the cholinergic amacrine cells are required for proper mosaic patterning.

Development of starburst cholinergic amacrine cells in the retina of Tupaia belangeri.

"Starburst" cholinergic amacrines specify the response of direction-selective ganglion cells to image motion. Here, development of cholinergic amacrines was studied in the tree shrew Tupaia belangeri (Scandentia) by immunohistochemistry with antibodies against choline acetyltransferase (ChAT) and neurofilament proteins. Starburst amacrines expressed ChAT much earlier than previously thought. From embryonic day 34 (E34) onward, orthotopic and displaced subpopulations segregated from a single cluster of immunoreactive precursor cells. Orthotopic starburst amacrines rapidly took up positions in the inner nuclear layer. Displaced starburst amacrines were first arranged in a monocellular row in the inner plexiform layer, and, with a delay of 1 week, they descended to the ganglion cell layer. Conversely, dendritic stratification of displaced amacrines slightly preceded that of orthotopic ones. Starburst amacrines expressed the medium-molecular-weight neurofilament protein (NF-M) from E34 to postnatal day 11 (P11) and coexpressed alpha-internexin from E36.5 to P11. Consequently, neurofilaments composed of alpha-internexin and NF-M may stabilize developing dendrites of starburst amacrines. During the first 2 postnatal weeks, subpopulations of anti-NF-M-labeled ganglion cells costratified with the preexisting dendritic strata of starburst amacrines in the ON sublamina, OFF sublamina, or both. Hence, anti-NF-M-labeled ganglion cells may include direction-selective ones. Thereafter, NF-M and alpha-internexin proteins disappeared from starburst amacrines, and NF-M immunoreactivity was lost in the dendrites of ganglion cells. Our findings suggest that NF-M and alpha-internexin are important for starburst amacrines and ganglion cells to recognize each other and, thus, contribute to the formation of early developing retinal circuits in the inner plexiform layer.

Vulnerability of dopaminergic amacrine cells and optic nerve myelination to prenatal endotoxin exposure.

PURPOSE: Intrauterine infection has been linked to preterm delivery and neurologic injury. The purpose of this study was to investigate the effects of fetal inflammation induced by exposure to endotoxin on the structure and neurochemistry of the retina and optic nerve. METHODS: The bacterial endotoxin, lipopolysaccharide (LPS), was administered to fetal sheep at approximately 0.65 of the approximately 147-day gestation period via repeated bolus doses (1 microg/kg per day) over 5 days, with fetal retinas and optic nerves assessed 10 days after the first LPS exposure. RESULTS: In the retina, the total number of tyrosine hydroxylase immunoreactive (TH-IR), dopaminergic amacrine cells was reduced (P < 0.05) in LPS-exposed compared with control fetuses. There was no difference in the number of ChAT-, substance P-, or NADPH-d-positive amacrine cells. The total number of myelinated axons in the optic nerve was not different (P > 0.05) between groups; however, the myelin sheath was thinner (P < 0.05) in LPS-exposed fetuses. CONCLUSIONS: Prenatal exposure to repeated doses of endotoxin results in alterations to the retina and optic nerve with specific effects on dopaminergic neurons and myelination, respectively. These findings could have implications for visual function.

Ptf1a determines horizontal and amacrine cell fates during mouse retinal development.

The vertebrate neural retina comprises six classes of neurons and one class of glial cells, all derived from a population of multipotent progenitors. There is little information on the molecular mechanisms governing the specification of cell type identity from multipotent progenitors in the developing retina. We report that Ptf1a, a basic-helix-loop-helix (bHLH) transcription factor, is transiently expressed by post-mitotic precursors in the developing mouse retina. Recombination-based lineage tracing analysis in vivo revealed that Ptf1a expression marks retinal precursors with competence to exclusively produce horizontal and amacrine neurons. Inactivation of Ptf1a leads to a fate-switch in these precursors that causes them to adopt a ganglion cell fate. This mis-specification of neurons results in a complete loss of horizontal cells, a profound decrease of amacrine cells and an increase in ganglion cells. Furthermore, we identify Ptf1a as a primary downstream target for Foxn4, a forkhead transcription factor involved in the genesis of horizontal and amacrine neurons. These data, together with the previous findings on Foxn4, provide a model in which the Foxn4-Ptf1a pathway plays a central role in directing the differentiation of retinal progenitors towards horizontal and amacrine cell fates.

Knock-down of GFRalpha4 expression by RNA interference affects the development of retinal cell types in three-dimensional histiotypic retinal spheres.

PURPOSE: To determine the role of glial cell line-derived neurotropic factor family receptor alpha 4 (GFRalpha4) during retinogenesis in a three-dimensional histiotypic in vitro model of the embryonic chicken retina. METHODS: Retinal spheres were cultured from dissociated 6-day-old chicken retina under permanent rotation and transfected with GFRalpha4 siRNA at culture day 2. Alterations on proliferation, apoptosis, and differentiation were determined by semiquantitative RT-PCR, in situ hybridization, and immunohistochemistry after 24, 48, and 72 hours. RESULTS: In contrast to control cultures, retinal spheres transfected with GFRalpha4 siRNA showed reduced GFRalpha4 mRNA expression of only 38% after 24 hours, 3% after 48 hours, and 5% after 72 hours. Based on the suppression of GFRalpha4, a decline in proliferating cells from 10% to 4.8% even after 24 hours and a reduction of sphere size by up to 25% at later culture stages were observed. Moreover, the number of Pax 6-positive amacrine, ganglion, and horizontal cells was significantly decreased from 36% to 16% in GFRalpha4 siRNA-transfected retinal spheres 72 hours after transfection. Additionally, GFRalpha4 gene silencing affected the development of different types of photoreceptors, as revealed by a significant decrease of blue opsin mRNA expression from 29% to 2%, whereas green opsin mRNA and the number rho4D2-positive photoreceptors were significantly increased. CONCLUSIONS: These data showed for the first time that GFRalpha4 plays an essential role in regulating, at least in vitro, the development and differentiation of various cell types during retinogenesis.

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.

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.

Multiple requirements for Hes 1 during early eye formation.

During embryogenesis, multiple developmental processes are integrated through their precise temporal regulation. Hes1 is a transcriptional repressor that regulates the timing of mammalian retinal neurogenesis. However, roles for Hes1 in early eye development have not been well defined. Here, we show that Hes1 is expressed in the forming lens, optic vesicle, cup, and pigmented epithelium and is necessary for proper growth, morphogenesis, and differentiation of these tissues. Because Hes1 is required throughout the eye, we investigated its interaction with Pax6. Hes1-Pax6 double mutant embryos are eyeless suggesting these genes are coordinately required for initial morphogenesis and outgrowth of the optic vesicle. In Hes1 mutants, Math5 expression is precocious along with retinal ganglion cell, amacrine, and horizontal neuron formation. In contrast to apparent cooperativity between Pax6 and Hes1 during morphogenesis, each gene regulates Math5 and RGC genesis independently. Together, these studies demonstrate that Hes1, like Pax6, simultaneously regulates multiple developmental processes during optic development.

Immunocytochemical development of the guinea pig retina.

The aim of the present study was to establish the neurochemical profile of amacrine and horizontal cells during ontogeny in the guinea pig, a precocial species where significant retinal development occurs prenatally as opposed to altricial species where development largely occurs postnatally. The expression of neurochemical markers of horizontal cells and specific amacrine cell populations was investigated from 20 days of gestation (dg, term approximately 67 dg) to adulthood. Amacrine cell populations were identified immunohistochemically using antibodies to gamma-amino-butyric acid, cholineacetyltransferase, calbindin, calretinin, neuronal nitric oxide synthetase and tyrosine hydroxylase; horizontal cells were labelled with calbindin. All markers were present at 30 dg and had attained their mature (adult) laminar distribution and expression by 60 dg. Horizontal cells appeared in their final location at 30 dg with amacrine cell populations appearing in their final locations by 45 dg. Thus, in the guinea pig retina, the amacrine and horizontal cell populations investigated in this study are fully mature prior to birth.

Sonic hedgehog, secreted by amacrine cells, acts as a short-range signal to direct differentiation and lamination in the zebrafish retina.

Neurogenesis in the zebrafish retina occurs in several waves of differentiation. The first neurogenic wave generates ganglion cells and depends on hedgehog (hh) signaling activity. Using transgenic zebrafish embryos that express GFP under the control of the sonic hedgehog (shh) promoter, we imaged the differentiation wave in the retina and show that, in addition to the wave in the ganglion cell layer, shh expression also spreads in the inner nuclear layer. This second wave generates amacrine cells expressing shh, and although it overlaps temporally with the first wave, it does not depend on it, as it occurs in the absence of ganglion cells. We also show that differentiation of cell types found in the inner and outer nuclear layers, as well as lamination of the retina, depends on shh. By performing mosaic analysis, we demonstrate that Shh directs these events as a short-range signal within the neural retina.

Chronic placental insufficiency affects retinal development in the guinea pig.

PURPOSE: Very low birth weight (VLBW) and fetal growth restriction are associated with increased risks of long-term visual impairments, including alterations to contrast sensitivity, a parameter mediated in part by dopaminergic amacrine cells. This study was conducted to determine whether chronic placental insufficiency (CPI), sufficient to cause growth restriction, results in neurochemical alterations to retinal interneurons, specifically amacrine and horizontal cell populations near term. METHODS: CPI was induced just before midgestation (term approximately 67 days of gestation, dg) in guinea pigs through unilateral ligation of the uterine artery. Growth-restricted (GR, n = 32) and control (n = 29) fetuses were euthanized at 60 dg and retinas prepared for analysis of amacrine cell populations by using antibodies to calbindin, calretinin, cholineacetyltransferase (ChAT), gamma-amino-butyric acid (GABA), dopamine beta-hydroxylase (D beta H), tyrosine hydroxylase (TH, dopaminergic), and NADPH-diaphorase histochemistry (nitrergic). Calbindin immunoreactivity (IR) was also used to identify horizontal cells. HPLC was used to assess concentrations of catecholamines and Western blot analysis to detect total TH levels. RESULTS: In GR compared with control fetuses the total number of TH-IR amacrine (P < 0.02) and calbindin-IR horizontal (P < 0.05) cells was reduced; however, there were no differences in the number of the ChAT, calbindin, calretinin, GABAergic, or nitrergic amacrine cell populations. HPLC revealed a reduction in the concentration of dopamine (P < 0.05) and noradrenaline (P < 0.05), and Western blot analysis revealed a reduction in TH in the retinas of GR compared with control fetuses (P < 0.05). CONCLUSIONS: CPI results in alterations to specific populations of retinal neurons. Such effects could contribute to visual impairments reported for VLBW children.

Differentiation of ganglion cells and amacrine cells in the rat retina: correlation with expression of HuC/D and GAP-43 proteins.

In order to understand the development of retinal cells, we have studied the temporal expression of HuC/D protein in embryonic, postnatal and adult rat retina. During development and in the adult retina, practically all cell somata in the ganglion cell layer and the vast majority of conventional amacrine cells in the inner nuclear layer displayed HuC/D immunoreactivity. Most but not all ganglion cells expressed HuC/D at embryonic day 15, suggesting a delay between final mitosis and the initiation of HuC/D expression. Immunoreactivity for HuC/D was also evident in developing but not mature horizontal cells. Combined immunohistochemical visualization of HuC/D protein and the growth-associated protein (GAP-43) showed a distinct localization of GAP-43 in a specific compartment close to the somato-dendritic region of developing HuC/D-positive cell somata. The localization of GAP-43 immunoreactivity to a specific soma compartment became less evident during maturation. Immunoreactivity for HuC/D and GAP-43 was also discernible in horizontal cells at postnatal day 14. In the adult retina, most GAP-43 immunoreactivity was seen in the inner plexiform layer. Detailed analysis showed that HuC/D and GAP-43 expression is restricted to subsets of retinal neurons during development and in the mature retina. Thus, GAP-43 appears to be correlated with initial steps of differentiation and outgrowth of dendritic processes in HuC/D-positive ganglion and amacrine cells.

Expression of SNAP-25 during mammalian retinal development: thinking outside the synapse.

The SNARE complex is the core machinery required for vesicle fusion events. Numerous structural, functional, and genetic studies have led to a better understanding of mechanisms that regulate vesicle fusion events during neural development. Studies using the mammalian retina as a model system have increased our understanding of the dynamic patterns of expression of SNARE proteins. In particular, the SNARE complex protein SNAP-25 is expressed in a dynamic fashion during the development of cholinergic amacrine cells in a number of mammalian species. SNAP-25 is also likely to play a crucial role during the development of vertebrate photoreceptors. The integration of comparative studies examining SNARE proteins, such as SNAP-25, provides a powerful approach for the study of CNS development.

Differential postreceptor signaling events triggered by excitotoxic stimulation of different ionotropic glutamate receptors in retinal neurons.

The aim of this work was to investigate whether excitotoxicity induced by overstimulation of different ionotropic glutamate receptors could trigger different intracellular signaling cascades. Cultured chick neuronal retina cells, essentially amacrine-like, were particularly sensitive to the toxicity induced by non-NMDA glutamate receptor agonists. One hour stimulation with 100 microM kainate induced a reduction of cell viability of about 44%, as assessed by the MTT test 24 hr after stimulation. Kainate-induced toxicity was mediated through AMPA receptors. Glutamate (100 microM, 1 hr) reduced cell viability by 26%, essentially acting through N-methyl-D-aspartate receptors. Five hours after stimulation, neuronal retina cells had an apoptotic-like nuclear morphology. In retinal neurons, the excitotoxic stimulation, with either glutamate or kainate, induced a calcium-dependent enhancement of the DNA-binding activity of the activating protein-1 (AP-1) transcription factor, which was maximal 2 hr after stimulation. Glutamate induced a greater increase in the AP-1 DNA-binding activity than did kainate. Supershift assays using antibodies directed against different members of the Fos and Jun protein families showed that the AP-1 complex in retinal neurons includes proteins of the Fos family, namely, Fra-2, c-Jun, and Jun D. The DNA-binding activity of the nuclear factor-kappaB transcription factor was not significantly changed upon excitotoxic stimulation with any agonist. Stimulation of glutamate receptors with 100 microM kainate or 100 microM glutamate for 2 min was sufficient to induce the activation of the extracellular signal-regulated kinase (ERK). Inhibition of the ERK activation with the MEK inhibitors U 0126 and PD 98059 increased the toxicity induced by kainate but was without effect on the toxicity induced by glutamate. These results indicate that, although stimulation with both glutamate receptor agonists increased ERK phosphorylation, only kainate-induced ERK activation correlates with the activation of a survival signaling pathway. Our results suggest that, in chick embryo retinal neurons, the signaling pathways that mediate excitotoxic cell death and neuroprotection are stimulus specific.