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.

Brain fatty acid profiles and spatial learning in malnourished rats: effects of nutritional intervention.

The aim of the present study was to determine the effects of malnutrition and nutritional rehabilitation on learning and memory performance and brain fatty acid composition. Pregnant and lactating Wistar rats were either fed ad libitum on a commercial laboratory chow or a multideficient diet from north-eastern Brazil (regional basic diet; RBD). After weaning, RBD offspring either continued on the multideficient diet (malnourished group) or switched to a control diet (rehabilitated group), until day 70. There was no difference in the passive avoidance test among the experimental groups, but malnourished rats showed important deficits in performance of the Morris water maze which were improved in the rehabilitated group. The hippocampus and cerebellum of the malnourished rats showed important changes in fatty acid profile obtained by gas-liquid chromatography, but the rehabilitated group had decreased n-3 polyunsaturated fatty acids and an increase in the proportion of arachidonic acid. The data suggest that nutritional rehabilitation results in partial restoration of fatty acid profiles and cognitive performance.

[Electroretinographic study of chromatic vision]. / Estudo eletrorretinográfico de visão cromática.

PURPOSE: To describe the electroretinogram of the South-American opossum (Didelphis aurita) obtained by chromatic stimulus of specific wavelengths. The electroretinogram records voltage variations of retinal cells triggered by light stimulation. The electroretinogram represents the combination of electric activity of many different cells and varies according to retinal physiology and examination methods. METHODS: We recorded the electroretinogram of six animals in dark adaptation using chromatic Kodak Wratten filters, and recorded the spectral sensitivity to specific wavelengths in the spectrum of blue, green, yellow, orange and red light bands. RESULTS: The most consistent electrorretinographic results were obtained when the animals were stimulated by selective spectral bands instead of white light. These results are consistent with the absorbance curve of the opsins described in marsupial photoreceptors. Previous studies using microspectrophotometry of opsins and retinal immunohistochemistry suggested marsupial trichromacy. This morphologic knowledge has not before been physiologically demonstrated by electroretinographic methods. CONCLUSION: The South-American opossum has proven to be an interesting experimental animal for comparative visual physiology studies among other mammals, especially studies on phylogenetic of chromatic vision. The opossum represents a retinal model that superimposes both the photopic and scotopic systems; and the Didelphis genus shows few changes when compared to the fossils of the Pleocene period. Therefore the marsupial's visual system retrieves characteristics from ancient mammal evolution to the retinal patterns found in modern mammals.

Pituitary adenylate cyclase-activating polypeptide (PACAP) can act as determinant of the tyrosine hydroxylase phenotype of dopaminergic cells during retina development.

In the chick retina, dopaminergic cells are generated between embryonic days 3 and 7 (E3/E7). However, the expression of tyrosine hydroxylase (TH), the first enzyme in the catecholamine synthetic pathway, is only detected after E11/E12. During the interval comprising E7 to E12, signals conveyed by cAMP are important to determine the TH phenotype. The present study shows that pituitary adenylyl cyclase-activating polypeptide (PACAP), via cAMP, is a major endogenous component in defining the TH phenotype of retina dopaminergic cells during development. PACAP type 1 receptor and its mRNA were detected in retinas since E6. PACAP was also immunodetected in cells localized in the inner nuclear layer of retinas since E8. This peptide promoted greater than 10-fold increase in cAMP accumulation of retinas obtained from embryos since E8, an effect that was blocked by PACAP6-38 (PAC1 receptor antagonist). In cultured retina cells from E8 and E9, maintained for 6 days in vitro with 10 nM PACAP (for 5 days), the number of dopaminergic cells expressing tyrosine hydroxylase increased 2.4-fold. The cAMP analog, 8-Br-cAMP and 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor) also increased the number of tyrosine hydroxylase-positive cells by 4- to 6-fold. IBMX plus PACAP treatment resulted in 17-fold increase in the number of cells positive for tyrosine hydroxylase. Under this condition the amount of tyrosine hydroxylase expression, as detected by western blot analysis, was also increased. The protein kinase-A inhibitor, rp-cAMPS, significantly reduced the effect of PACAP. Our data show that this peptide is an important factor influencing the definition of the tyrosine hydroxylase phenotype of retina dopaminergic cells within a narrow window of development.

Cultured embryonic retina systems as a model for the study of underlying mechanisms of Toxoplasma gondii infection.

PURPOSE: Toxoplasma gondii, the most common cause of retinochoroiditis in humans, is an obligate intracellular protozoan parasite that depends on te host cell's microenvironment to proliferate. Because congenital infection is associated with a higher risk of ocular involvement than a postnatally acquired infection, this study was conducted to investigate the ability of Toxoplasma gondii to infect retinal tissue during development, when cellular environmental changes normally occur. METHODS: Retinas from 5- to 9-day-old chick embryos were used. Stationary cultures were prepared in 24-well cell culture dishes and maintained at 37 degrees C in DMEM plus 5% fetal bovine serum for 2 to 6 days. Then the wells were infected with 4 x 10(5) tachyzoites. Retina explants and aggregate cell cultures were maintained in DMEM under rotation at 37 degrees C. T. gondii proliferation was measured using [(3)H]-thymidine incorporation after 72 hours. Ornithine and arginine decarboxylase (ODC and ADC) activities were determined by measuring CO(2) production from [1-(14)C]-ornithine and [1-(14)C]-arginine, respectively. RESULTS: The proliferation of tachyzoites was high in dense, stationary cultures expressing elevated ODC and ADC activity. The addition of ODC or ADC inhibitors reduced T. gondii proliferation by approximately 20% to 40%. As for cultured retina cells, retina explants also allowed T. gondii proliferation whenever ODC activity was high. CONCLUSIONS: The data suggest a direct correlation between retinal polyamine biosynthesis and the proliferation of T. gondii, in agreement with the observation that individuals infected congenitally have a greater risk of development of toxoplasmic retinochoroiditis.

Differential immunodetection of L-DOPA decarboxylase and tyrosine hydroxylase in the vertebrate retina.

The immunocytochemical staining of L-DOPA decarboxylase (DDC) and tyrosine hydroxylase (TH) in cells of the developing avian retina and in cells of the retina of adult rats and opossum were compared. DDC was identified at embryonic day 8 in the chick, in cells in the inner nuclear layer (INL). At embryonic day 13, two types of DDC positive cells were observed; type 1, with the soma located in the innermost layer of the INL; and type 2, with the soma located two cell rows from the innermost part of the INL. Immunolabeling for DDC in the presumptive outer plexiform layer was more clearly defined at embryonic day 19 and at post-hatched day 7. Processes of DDC labeled cells extended into the inner plexiform layer, supporting the amacrine identity of these cells. Dot-blot analysis revealed that DDC could be detected at embryonic day 4. Confocal microscopy showed that at embryonic day 10, DDC positive cells, but not TH positive cells, were found. After embryonic day 13, cells immunolabeled for DDC and DDC plus TH were detected. The mean density of DDC positive cells quantified in whole-mounted chick retinas showed that in all stages the density of DDC positive cells exceeded that of TH positive cells by 10-13-fold. As for the avian retina, density of DDC positive cells in opossum and rat retinas exceeded the density of TH positive cells. In opossum, Müller fibers were also clearly labeled for DDC but not for TH. We propose the hypothesis that the dopamine synthesis in the developing avian retina as well as in the mature rat and opossum tissue is greater than would be expected from TH staining alone.

Coexistence of GAD-65 and GAD-67 with tyrosine hydroxylase and nitric oxide synthase in amacrine and interplexiform cells of the primate, Cebus apella.

The expression of glutamate decarboxylase forms, GAD-65 and GAD-67, in GABAergic cells was studied by immunocytochemistry in the retina of the New World monkey, Cebus apella. In the innermost rows of the inner nuclear layer (INL), somata that express GABA correspond to about 45% of the total number of cells in the central retina and about 25% on the periphery. Three subsets of GABAergic amacrine cells were identified along the horizontal meridian: about 5% express only GAD-65 and 40% GAD-67, and approximately 50% contain both forms of GAD. In the INL, GAD-65 immunoreactivity was detected in broad bands around strata 1, 3, and 5. GAD-67 immunoreactivity was observed throughout all strata. Somata that expressed GAD-67 exclusively stratified only in narrow bands around strata 2 and 4 of the INL and colocalized with beta2 and beta3 subunits of GABA-A receptor. Interplexiform and amacrine cells that express GABA also express tyrosine hydroxylase (TH) or nitric oxide synthase (NOS). GAD-67 colocalized with TH or NOS in presumed amacrine cells of the inner plexiform layer (IPL) and ganglion cell layer (GCL). GAD-65 was expressed in the TH- and NOS-immunoreactive interplexiform and amacrine cells, respectively. Different from what has been described in other mammals, TH and NOS were coexpressed in some neurons, indicating a partial overlap in retinal cell populations containing dopamine or nitric oxide in this primate.

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.

Estudo eletrorretinográfico de visão cromática / Electroretinographic study of chromatic vision

OBJETIVO: O objetivo deste estudo é descrever o traçado eletrorretinográfico no gambá sul-americano (Didelphis aurita) obtido com estímulo cromático de comprimento de onda seletivo. O eletrorretinograma é o registro das variações de voltagem nas células retinianas, desencadeadas por estímulo luminoso. O eletrorretinograma representa a atividade elétrica combinada de diferentes células, e sofre variações dependendo da fisiologia retiniana e do método de exame. MÉTODOS: Foram registrados os eletrorretinogramas de seis animais em adaptação ao escuro utilizando filtros cromáticos Kodak Wratten®, e registrada a sensibilidade espectral para comprimentos de onda específicos nas faixas de cores do azul, verde, amarelo, laranja e vermelho. RESULTADOS: Os resultados eletrorretinográficos mais consistentes foram obtidos quando o animal foi estimulado por faixas espectrais seletivas, ao invés de luz branca; e são consistentes com a curva de absorbância das opsinas descritas em fotorreceptores de marsupiais. Estudos prévios sugeriram a tricromacia dos marsupiais por microespectrofotometria de opsinas e imuno-histoquímica de retina. Esse fundamento morfológico não tinha demonstração fisiológica eletrorretinográfica, até este estudo. CONCLUSÃO: O gambá sul-americano tem se mostrado interessante como animal experimental no estudo comparativo da fisiologia visual em mamíferos, especialmente no estudo filogenético da visão cromática. Os marsupiais apresentam um modelo retiniano que superpõe os sistemas fotópico e escotópico; e o gênero Didelphis conserva características encontradas em fósseis do período pleoceno. Portanto, o sistema visual de um marsupial resgata características dos primórdios da evolução dos mamíferos, até o desenvolvimento dos padrões retinianos modernos.

PURPOSE: To describe the electroretinogram of the South-American opossum (Didelphis aurita) obtained by chromatic stimulus of specific wavelengths. The electroretinogram records voltage variations of retinal cells triggered by light stimulation. The electroretinogram represents the combination of electric activity of many different cells and varies according to retinal physiology and examination methods. METHODS: We recorded the electroretinogram of six animals in dark adaptation using chromatic Kodak Wratten® filters, and recorded the spectral sensitivity to specific wavelengths in the spectrum of blue, green, yellow, orange and red light bands. RESULTS: The most consistent electrorretinographic results were obtained when the animals were stimulated by selective spectral bands instead of white light. These results are consistent with the absorbance curve of the opsins described in marsupial photoreceptors. Previous studies using microspectrophotometry of opsins and retinal immunohistochemistry suggested marsupial trichromacy. This morphologic knowledge has not before been physiologically demonstrated by electroretinographic methods. CONCLUSION: The South-American opossum has proven to be an interesting experimental animal for comparative visual physiology studies among other mammals, especially studies on phylogenetic of chromatic vision. The opossum represents a retinal model that superimposes both the photopic and scotopic systems; and the Didelphis genus shows few changes when compared to the fossils of the Pleocene period. Therefore the marsupial's visual system retrieves characteristics from ancient mammal evolution to the retinal patterns found in modern mammals.

Watery and dark axons in Wallerian degeneration of the opossum's optic nerve: different patterns of cytoskeletal breakdown?

In this paper we report a qualitative morphological analysis of Wallerian degeneration in a marsupial. Right optic nerves of opossums Didelphis marsupialis were crushed with a fine forceps and after 24, 48, 72, 96 and 168 hours the animals were anaesthetized and perfused with fixative. The optic nerves were immersed in fixative and processed for routine transmission electron microscopy. Among the early alterations typical of axonal degeneration, we observed nerve fibers with focal degeneration of the axoplasmic cytoskeleton, watery degeneration and dark degeneration, the latter being prevalent at 168 hours after crush. Our results point to a gradual disintegration of the axoplasmic cytoskeleton, opposed to the previous view of an "all-or-nothing'' process (Griffin et al 1995). We also report that, due to an unknown mechanism, fibers show either a dark or watery pattern of axonal degeneration, as observed in axon profiles. We also observed fibers undergoing early myelin breakdown in the absence of axonal alterations

The opossum photoreceptors: a model for evolutionary trends in early mammalian retina

The topography and spectral characteristics of mammalian photoreceptors correlate with both, the present ecological demands and the evolutionary history. The South American Opossum is a marsupial mammal with unspecialized habitus and crepuscular lifestyle. A sparse population of cones (max. = 3000/mm2) can be differentiated into four subtypes by morphological, topographical and immunocytochemical criteria. In spite of this unusual diversity the cone types can be split into two functional groups: The population of single cones labeled by antibody OS-2 for short wavelenght sensitive pigments was ubiquitous but at very low densities (200/mm2). The single cones labeled by antibody (COS-1) against long wavelength sensitive pigments constitute the dominant population in the area centralis (2300/mm2). These two single cone types correlate with the pair typically present in placental mammals. Discrimination of spatial and color contrast may be provided by this "modern" set. The COS-1 labeled double and single cones bearing an oil droplet, display a different pattern by being restricted to the inferior (non-tapetal) half of the retina (max = 800/mm2). This additional set of cones with oil droplets and long wavelength pigments is a conservative feature of the opossum retina and other marsupials. As an accessory cone system it is possibly providing enhanced sensitivity at mesopic conditions. During the early evolution of nocturnal mammals with its prominent expansion of rod vision these cone types were conserved but then were lost in placental mammals. Thus the unique features of mammalian are the result of two evolutionary steps: first a reduction of cone based vision, followed by a secondary differentiation of photopic vision and behaviour relying on the remaining set of cones.

The retinal distribution of ganglion cells with crossed and uncrossed projections and the visual field representation in the opossum

The retinal distribution of ganglion cells with crossed and uncrossed projections in the South American opossum, Didelphis marsupialis, was revealed by delivering HRP to one optic tract or to retinal targets of one hemisphere. The cells with uncrossed projections are restricted to the temporal retina, comprising 1/3 of the total retinal area, with a sharp transition at the naso-temporal boundary. Besides being distributed over the nasal 2/3 of the retina, cells with crossed projections are intermingled with those with uncrossed projections over the entire temporal retina. Quantitative analysis about the representation of the horizontal meridian on four specimens revealed that the maximun density of cells with uncrossed projection is on the average located at 3.2 mm (SD = 0.21), i.e. 34.8 deg, temporal to the optic disk, falling to 10% at 2.1 mm (SD = 0.14) or 22.8 deg. On the otherhand, the peak for cells with crossed projections is more nasally placed at 1.8 mm (SD=0.18), i.e. 19.6 deg. Between these two maxima, the site where in the densities of cells with crossed and uncrossed projections are about equal is on the average about 2.7 mm (SD = 0.25) form the optic disk, i.e. 29.3 deg. This estimate supports the hypothesis that the retinal itersection of the vertical meridian lies within the region of split representation of crossed and uncrossed ganglion cells. In addition, it was observed that the opossum's retina has a large contingent of cells with uncrossed projections temporal to an eccentricity of 2.7 mm from the optic disk, where it represents roughly 2/3 of the ganglion cells. These data corroborate the relevance of the opossum as a non-primate model for visual work

Neurotransmitter release in aggregate cultures of chick embryo retina cells

The study of neurotransmitter release using aggregate cell cultures has been limited by the fact that cell aggregates are obtained only when cells are maintained in suspension with rotatory agitation. In this report we describe a simple and easy method that uses aggregate cell cultures to study the release of neurotransmitters. The results demonstrate that this relatively simple technique can be of great value to address the problem of neurotransmitter release and study the mechanisms of action of natural and synthetic compounds on the differentiation of functional synapses in the CNS.