Cell-specific effects of RB or RB/p107 loss on retinal development implicate an intrinsically death-resistant cell-of-origin in retinoblastoma.
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Retinogenesis involves expansion of pluripotent progenitors, specification of postmitotic precursors, and terminal differentiation. Rb or Rb/p107 loss causes retinoblastoma in humans or mice, respectively. One model suggests that Rb- or Rb/p107-deficient retinal precursors have infinite proliferative capacity but are death-prone and must acquire an antiapoptotic mutation. Indeed, we show that Rb/p107 loss does not affect progenitor proliferation or precursor specification, but perturbs cell cycle exit in all seven retinal precursors. However, three precursors survive Rb/p107-loss and stop proliferating following terminal differentiation. Tumors arise from precursors that escape this delayed growth arrest. Thus, retinoblastoma arises from a precursor that has extended, not infinite, proliferative capacity, and is intrinsically death-resistant, not death-prone. We suggest that additional lesions common in retinoblastoma overcome growth arrest, not apoptosis. (+info)
Image defocus modulates activity of bipolar and amacrine cells in macaque retina.
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PURPOSE: To demonstrate regulation of ocular growth and refractive development by image quality and processing in the retina in higher primates. Focus-sensitive retinal neurons were labeled with immunocytochemical markers after briefly altering image quality in infant monkeys. METHODS: Six rhesus monkeys (Macaca mulatta) 20- to 30-days-old were fitted with goggles after 6 hours in the light. Three wore a +3 D lens and three a diffuser on the treated eye; contralateral control eyes wore plano lenses matched in transmission to the goggles on treated eyes. After 30, 60, or 240 minutes exposure, the animals were killed, the eyes opened and fixed in 4% formaldehyde, and cryosections labeled with antibodies to inducible activity markers (transcription factors Egr-1 and Fra-2) and type-specific amacrine cell markers. Labeled cells were identified and counted in a fluorescence microscope, and the spatial density of activity-labeled nuclei and the frequency of activity-labeling of specific amacrine cells were determined, without knowing treated eye or duration. RESULTS: Focus-sensitive immunoreactivity was demonstrated for Egr-1 and Fra-2 in a GAD65-immunoreactive (IR) subpopulation of GABAergic amacrine cells, and for Egr-1 alone in PKC alpha-, 115A10-, and CD15-IR ON-bipolar cells. Activity of ON-bipolar and GABAergic amacrine cells, as indicated by Egr-1 induction, was stimulated more by in-focus or myopically-defocused images than by hyperopically-defocused or diffusely blurred images, regardless of exposure duration. CONCLUSIONS: This was the first evidence of focus-dependent activation of bipolar as well as amacrine cells in a primate retina. Focus-sensitive neurons are candidates for roles in vision-dependent regulation of eye growth. (+info)
Chronic placental insufficiency affects retinal development in the guinea pig.
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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. (+info)
Sonic hedgehog, secreted by amacrine cells, acts as a short-range signal to direct differentiation and lamination in the zebrafish retina.
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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. (+info)
Identification of retinal neurons in a regressive rodent eye (the naked mole-rat).
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The retina consists of many parallel circuits designed to maximize the gathering of important information from the environment. Each of these circuits is comprised of a number of different cell types combined in modules that tile the retina. To a subterranean animal, vision is of relatively less importance. Knowledge of how circuits and their elements are altered in response to the subterranean environment is useful both in understanding processes of regressive evolution and in retinal processing itself. We examined common cell types in the retina of the naked mole-rat, Heterocephalus glaber with immunocytochemical markers and retrograde staining of ganglion cells from optic nerve injections. The stains used show that the naked mole-rat eye has retained multiple ganglion cell types, 1-2 types of horizontal cell, rod bipolar and multiple types of cone bipolar cells, and several types of common amacrine cells. However, no labeling was found with antibodies to the dopamine-synthesizing enzyme, tyrosine hydroxylase. Although most of the well-characterized mammalian cell types are present in the regressive mole-rat eye, their structural organization is considerably less regular than in more sighted mammals. We found less precision of depth of stratification in the inner plexiform layer and also less precision in their lateral coverage of the retina. The results suggest that image formation is not very important in these animals, but that circuits beyond those required for circadian entrainment remain in place. (+info)
A unique role for Kv3 voltage-gated potassium channels in starburst amacrine cell signaling in mouse retina.
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Direction-selective retinal ganglion cells show an increased activity evoked by light stimuli moving in the preferred direction. This selectivity is governed by direction-selective inhibition from starburst amacrine cells occurring during stimulus movement in the opposite or null direction. To understand the intrinsic membrane properties of starburst cells responsible for direction-selective GABA release, we performed whole-cell recordings from starburst cells in mouse retina. Voltage-clamp recordings revealed prominent voltage-dependent K(+) currents. The currents were mostly blocked by 1 mm TEA, activated rapidly at voltages more positive than -20 mV, and deactivated quickly, properties reminiscent of the currents carried by the Kv3 subfamily of K+ channels. Immunoblots confirmed the presence of Kv3.1 and Kv3.2 proteins in retina and immunohistochemistry revealed their expression in starburst cell somata and dendrites. The Kv3-like current in starburst cells was absent in Kv3.1-Kv3.2 knock-out mice. Current-clamp recordings showed that the fast activation of the Kv3 channels provides a voltage-dependent shunt that limits depolarization of the soma to potentials more positive than -20 mV. This provides a mechanism likely to contribute to the electrical isolation of individual starburst cell dendrites, a property thought essential for direction selectivity. This function of Kv3 channels differs from that in other neurons where they facilitate high-frequency repetitive firing. Moreover, we found a gradient in the intensity of Kv3.1b immunolabeling favoring proximal regions of starburst cells. We hypothesize that this Kv3 channel gradient contributes to the preference for centrifugal signal flow in dendrites underlying direction-selective GABA release from starburst amacrine cells (+info)
Vesicular glutamate transporter 3 expression identifies glutamatergic amacrine cells in the rodent retina.
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Synaptic transmission from glutamatergic neurons requires vesicular glutamate transporters (VGLUTs) to concentrate cytosolic glutamate in synaptic vesicles. In retina, glutamatergic photoreceptors and bipolar cells exclusively express the VGLUT1 isoform, whereas ganglion cells express VGLUT2. Surprisingly, the recently identified VGLUT3 isoform was found in presumed amacrine cells, generally considered to be inhibitory interneurons. To investigate the synaptic machinery and conceivable secondary neurotransmitter composition of VGLUT3 cells, and to determine a potential functional role, we further investigated these putative glutamatergic amacrine cells in adult and developing rodent retina. Reverse transcriptase-PCR substantiated VGLUT3 expression in mouse retina. VGLUT3 cells did not immunostain for ganglion or bipolar cell markers, providing evidence that they are amacrine cells. VGLUT3 colocalized with synaptic vesicle markers, and electron microscopy showed that VGLUT3 immunostained synaptic vesicles. VGLUT3 cells were not immunoreactive for amacrine cell markers gamma-aminobutyric acid, choline acetyltransferase, calretinin, or tyrosine hydroxylase, although they immunostain for glycine. VGLUT3 processes made synaptic contact with ganglion cell dendrites, suggesting input onto these cells. VGLUT3 immunostaining was closely associated with the metabotropic glutamate receptor 4, which is consistent with glutamatergic synaptic exocytosis by these cells. In the maturing mouse retina, Western blots showed VGLUT3 expression at postnatal day 7/8 (P7/8). VGLUT3 immunostaining in retinal sections was first observed at P8, achieving an adult pattern at P12. Thus, VGLUT3 function commences around the same time as VGLUT1-mediated glutamatergic transmission from bipolar cells. Furthermore, a subset of VGLUT3 cells expressed the circadian clock gene period 1, implicating VGLUT3 cells as part of the light-entrainable retina-based circadian system. (+info)
Involvement of brain-derived neurotrophic factor in early retinal neuropathy of streptozotocin-induced diabetes in rats: therapeutic potential of brain-derived neurotrophic factor for dopaminergic amacrine cells.
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Although neurotrophins have been assessed as candidate therapeutic agents for neural complications of diabetes, their involvement in diabetic retinopathy has not been fully characterized. We found that the protein and mRNA levels of brain-derived neurotrophic factor (BDNF) in streptozotocin-induced diabetic rat retinas were reduced to 49% (P < 0.005) and 74% (P < 0.05), respectively, of those of normal control animals. In addition, dopaminergic amacrine cells appeared to be degenerating in the diabetic rat retinas, as revealed by tyrosine hydroxylase (TH) immunoreactivity. Overall TH protein levels in the retina were decreased to one-half that of controls (P < 0.01), reflecting reductions in the density of dopaminergic amacrine cells and the intensity of TH immunoreactivity within them. To confirm the neuropathological implications of BDNF reduction, we administered BDNF protein into the vitreous cavities of diabetic rats. Intraocular administration of BDNF rescued dopaminergic amacrine cells from neurodegeneration and counteracted the downregulation of TH expression, demonstrating its therapeutic potential. These findings suggest that the early retinal neuropathy of diabetes involves the reduced expression of BDNF and can be ameliorated by an exogenous supply of this neurotrophin. (+info)