Jan Nora Hokoç

A comparative study of neurogenesis in the retinal ciliary marginal zone of homeothermic vertebrates

The retina of many fish and amphibians grows throughout life, roughly matching the overall growth of the animal. The new retinal cells are continually added at the anterior margin of the retina, in a circumferential zone of cells, known as the ciliary marginal zone, or CMZ. Recently, Fischer and Reh [Dev. Biol. 220 (2000) 197] have found that new neurons are added to the retina of the chicken via proliferation and subsequent differentiation of neurons and glia at the retinal margin in a zone highly reminiscent of the CMZ of lower vertebrates. In addition, other groups have reported that putative retinal stem cells could be isolated from the ciliary margin of the adult mouse. In light of these findings, we have re-investigated the eyes of three additional species to determine whether other homeothermic vertebrates also possess CMZ cells and whether we could detect evidence for addition of neurons at the retinal margin in mature animals. We examined one additional avian species, the quail, one marsupial, the opposum, and one mammal, the mouse. We find that the CMZ cells have been gradually diminished during vertebrate evolution. The quail has a reduced CMZ as compared to the chicken, while the opposum has only a few cells likely related to the CMZ and we failed to find evidence of CMZ cells at the margin of the mouse retina.

Thyroid hormone action is required for normal cone opsin expression during mouse retinal development.

PURPOSE The expression of S- and M-opsins in the murine retina is altered in different transgenic mouse models with mutations in the thyroid hormone receptor (TR)-beta gene, demonstrating an important role of thyroid hormone (TH) in retinal development. METHODS The spatial expression of S- and M-opsin was compared in congenital hypothyroidism and in two different TR mutant mouse models. One mouse model contains a ligand-binding mutation that abolishes TH binding and results in constitutive binding to nuclear corepressors. The second model contains a mutation that blocks binding of coactivators to the AF-2 domain without affecting TH binding. RESULTS Hypothyroid newborn mice showed an increase in S-opsin expression that was completely independent of the genotype. Concerning M-opsin expression, hypothyroidism caused a significant decrease (P < 0.01) only in wild-type animals. When TRbeta1 and -beta2 were T3-binding defective, the pattern of opsin expression was similar to TRbeta ablation, showing increased S-opsin expression in the dorsal retina and no expression of M-opsin in the entire retina. In an unexpected finding, immunostaining for both opsins was detected when both subtypes of TRbeta were mutated in the helix 12 AF-2 domain. CONCLUSIONS The results show, for the first time, that the expression of S- and M-opsin is dependent on normal thyroid hormone levels during development.

Photoreceptors in a primitive mammal, the South American opossum, Didelphis marsupialis aurita: Characterization with anti-opsin immunolabeling

The retinas of placental mammals appear to lack the large number and morphological diversity of cone subtypes found in diurnal reptiles. We have now studied the photoreceptor layer of a South American marsupial (Didelphis marsupialis aurita) by peanut agglutinin labeling of the cone sheath and by labeling of cone outer segments with monoclonal anti-visual pigment antibodies that have been proven to consistently label middle-to-long wavelength (COS-1) and short-wavelength (OS-2) cone subpopulations in placental mammals. Besides a dominant rod population (max. = 400,000/mm2) four subtypes of cones (max. = 3000/mm2) were identified. The outer segments of three cone subtypes were labeled by COS-1: a double cone with a principal cone containing a colorless oil droplet, a single cone with oil droplet, and another single cone. A second group of single cones lacking oil droplets was labeled by OS-2 antibody. The topography of these cone subtypes showed striking anisotropies. The COS-1 labeled single cones without oil droplets were found all over the retina and constituted the dominant population in the area centralis located in the temporal quadrant of the upper, tapetal hemisphere. The population of OS-2 labeled cones was also ubiquitous although slightly higher in the upper hemisphere (200/mm2). The COS-1 labeled cones bearing an oil droplet, including the principal member of double cones, were concentrated (800/mm2) in the inferior, non-tapetal half of the retina. The two spectral types of single cones resemble those of dichromatic photopic systems in most placental mammals. The additional set of COS-1 labeled cones is a distinct marsupial feature. The presence of oil droplets in this cone subpopulation, its absence in the area centralis, and the correlation with the non-tapetal inferior hemisphere suggest a functional specialization, possibly for mesopic conditions. Thus, sauropsid features have been retained but probably with a modified function.

GABAergic system in the developing mammalian retina: dual sources of GABA at early stages of postnatal development

In the present work, we have characterized the maturation of the GABAergic system in mammalian retina. Immunoreactivity for GABA, GAD (glutamic acid decarboxylase, EC 4.1.1.15) ‐65 and ‐67 in the adult rat retina was localized in cells in the inner nuclear and ganglion cell layers. This pattern was established around postnatal day 8 and included transient GABA and GAD‐67 expression in horizontal cells. GAD activity was very low at P1 and P4, increasing after P8, reaching maximal activity by P21 and decreasing to attain adult values by P30. GABA content was approximately constant from P1 to P13, increasing thereafter to reach adult levels. GAD protein content increased progressively with postnatal development and the two isoforms could be distinguished at P8.

A schematic eye for the opossum

Abstract Schematic values were determined for the elements of the dioptric system of the eye of a marsupial. D. marsupialis aurita . Based on measurements on excised eyes a schematic eye was developed for this species. The following aspects of the opossums eye are discussed: real, entrance and exit pupils, retinal illumination and uniocular optical field. The extent of the retinal visual field was determined and the resulting cyclopic field established. The position of the blind and optic axes in space were indirectly determined by correlating the posture of the head under experimental and normal conditions, and the horizontal meridian was found to lie 4° below the blind axis.

Histogenesis and topographical distribution of tyrosine hydroxylase immunoreactive amacrine cells in the developing chick retina.

There is a delay from the time when amacrine cells are generated to the time when the dopaminergic phenotype is first expressed, in the chick retina. In order to determine the birthdate of amacrine cells expressing the tyrosine hydroxylase (TH) phenotype, we combined autoradiography of [3H]thymidine incorporated into dividing cells with the immunocytochemical method for TH in mature retinas. We also investigated the morphogenesis and the topographical distribution of dopaminergic amacrine cells using radial and horizontal sections of the chick retina. Although TH immunoreactivity was first detected at E12, the morphological pattern of TH-immunoreactive (TH-IR) amacrine cells started to be defined at E16, with an increasing arborization complexity until hatching. The topographical distribution of dopaminergic cells revealed that TH-IR neurons were predominantly concentrated in the dorsal retina of E13 and E14 embryos. At E18 and PH2 the distribution of dopaminergic cells was uniform throughout the retina. Autoradiography of [3H]thymidine incorporated association with TH immunocytochemistry showed that dopaminergic amacrine cells are generated during a discrete period (E3 through E7) of amacrinogenesis that occurs from E3 to E9. Therefore, a delay of days between histogenesis of dopaminergic amacrine cells and their differentiation is observed.

Photoreceptor topography of the retina in the New World monkey Cebus apella.

The number and topographical distribution of photoreceptors was studied in whole-mounted retinas of Cebus apella. It was estimated a total of 48 million rods and 3.8 million cones. The average peak foveal cone density and the Nyquist Limit at the foveola were estimated as 169, 127 cells/mm(2) and 46.77+/-7.98 cyc/deg, respectively. A cone-enriched rim was found near the ora serrata, more noticeable in the nasal retina. Rod distribution was asymmetrical along horizontal and vertical meridians with a higher density in the dorsal retina. The rod/cone ratio was variable and asymmetrical along both meridians.

L-DOPA supply to the neuro retina activates dopaminergic communication at the early stages of embryonic development

DOPA decarboxylase (DDC; aromatic‐l‐amino acid decarboxylase; EC 4.1.1.28) is absent in retinas from 6‐day‐old chicken embryos (E6) but is expressed in retina of E8 embryos, in the presumptive outer plexiform layer. Thereafter, DDC appears in cell bodies of presumptive amacrine cells. The dopamine (DA) content of E9/10 and E15/16 retinas, pre‐incubated with l‐DOPA for 1 h, increased 250‐ and 600‐fold, respectively, showing that DDC is active since early in development. Intercellular communication, measured by endogenous cyclic AMP accumulation, was observed when retinas from E9/10 to E15/16 were pre‐incubated for 1 h with 1 mm l‐DOPA, washed and followed by incubation in the presence of 0.5 mm 3‐isobutyl‐1‐methylxanthine, a phosphodiesterase inhibitor. Cyclic AMP accumulation was prevented when pre‐incubation with l‐DOPA was carried out in the presence of carbidopa. Moreover, the accumulation of cyclic AMP was inhibited by SCH 23390 (2 µm). The incubation of retinas in medium previously conditioned by retina‐pigmented epithelium (RPE) also increased its cyclic AMP content with the characteristics described for l‐DOPA. Our results show that dopaminergic communication takes place in the embryonic retina, before tyrosine hydroxylase expression, provided l‐DOPA is supplied to the tissue. It also shows that RPE is a potential source of l‐DOPA early in development.

Calpain inhibitor 2 prevents axonal degeneration of opossum optic nerve fibers

The ultrastructural change that characterizes the onset of Wallerian degeneration is the disintegration of axoplasmic microtubules and neurofilaments, which are converted into an amorphous and granular material, followed by myelin breakdown. The mechanism underlying such processes is an increase in the amount of intracellular calcium, leading to activation of proteases called calpains. The aim of this study was to evaluate by quantitative ultrastructural analysis whether nerve fibers can be preserved by the use of an exogenous inhibitor of these proteases (calpain inhibitor‐2, Mu‐F‐hF‐FMK), after optic nerve crush. For that, the left optic nerves of opossums, Didelphis aurita, were crushed with the aid of a fine forceps, and half of them received a calpain inhibitor mixed with Elvax resin. Ninety‐six hours after the lesion, the animals were reanesthetized and transcardially perfused, and the optic nerves were removed, the right ones being used as normal nerves. Afterward, the optic nerves were dissected and processed for routine transmission electron microscopy and quantitative and statistical analysis. The results of this analysis showed that the group that received the calpain inhibitor presented a reduction of astrogliosis, maintaining the optic nerve structure in an organized state; a significant decrease in the number of degenerating fibers; and a significant increase in the number of fibers with preserved cytoskeleton and preservation of axonal and myelin area and integrity, reducing the enlargement and edema of the axon. In conclusion, our findings suggest that calpain inhibitor is able to provide neuroprotection of the central nervous system fibers after a crush lesion.

Development of NADPH-Diaphorase in the Avian Retina: Regulation by Calcium Ions and Relation to Nitric Oxide Synthase

Abstract: Nitric oxide plays an important role as an intercellular messenger in the CNS. In the present work we measured NADPH‐diaphorase activity, which is considered to be a marker of cells producing nitric oxide, in homogenates of the developing chick retina. The enzyme activity can be detected beginning in 8‐day‐old embryonic retinas with no further quantitative variations throughout development. Arginine analogues inhibit ∼65% of the activity in embryonic retinas and 50% in posthatched retinas. The enzyme is stimulated 50% by 2 mM calcium chloride in retinas from 8 to 14 embryonic days, but this effect decreases to 20% in 17‐day embryonic retinas and practically disappears in posthatched animals. The stimulation by calcium is completely blocked by arginine analogues. The decrease in enzyme activity at posthatched retinas is not due to stimulation by endogenous calcium or the presence of insufficient amounts of calmodulin, because addition of EGTA or calmodulin, respectively, did not restore the stimulation to levels observed at embryonic stages. Inhibition of NADPH‐diaphorase activity by NG‐nitro‐l‐arginine or l‐NG‐(iminoethyl)ornithine is concentration dependent with IC50 values of ∼1 mM at all stages studied. However, in the presence of calcium, the inhibition by both analogues is shifted to the left and is apparently biphasic at all developmental stages, including in posthatched animals, with IC50 values in the low micromolar range. NADPH‐diaphorase was also detected by histochemistry in specific groups of cells in the early embryonic retina and in subsets of amacrine and ganglion cells, as well as in photoreceptors, in more developed retinas. The results indicate that different isoforms of nitric oxide synthase are present in the chick retina and that a calcium‐dependent isoform is predominant in early periods of development.