Chronic nutritional restriction of omega-3 fatty acids induces a pro-inflammatory profile during the development of the rat visual system.

Modern western diets have been associated with a reduced proportion of dietary omega-3 fatty acids leading to decreased levels of DHA (docosahexaenoic acid) in the brain. Low DHA content has been associated with altered development of visual acuity in infants and also with an altered time course of synapse elimination and plasticity in subcortical visual nuclei in rodents. Microglia has an active role in normal developmental processes such as circuitry refinement and plasticity, and its activation status can be modulated by omega-3 (ω3) and omega-6 (ω6) essential fatty acids. In the present study, we investigated the impact of dietary restriction of DHA (ω3-), through the chronic administration of a coconut-based diet as the only fat source. This dietary protocol resulted in a reduction in DHA content in the retina and superior colliculus (SC) and in a neuroinflammatory outcome during the development of the rodent visual system. The ω3- group showed changes in microglial morphology in the retina and SC and a corresponding altered pattern of pro-inflammatory cytokine expression. Early and late fish oil protocols supplementation were able to restore DHA levels. The early supplementation also decreased neuroinflammatory markers in the visual system. The present study indicates that a chronic dietary restriction of omega-3 fatty acids and the resulting deficits in DHA content, commonly observed in Western diets, interferes with the microglial profile leading to an inflamed microenvironment which may underlie a disruption of synapse elimination, altered topographical organization, abnormal plasticity, and duration of critical periods during brain development.

Age-related increase of let-7 family microRNA in rat retina and vitreous.

Vitreous alterations occur from early stages and continue through the normal aging, with gradual lamellae formation and the appearance of liquefied spaces, which eventually leads to complications, such as retinal tear, retinal detachment, and intravitreal hemorrhage. The aim of the present study was to investigate the expression of let-7 miRNA family in the vitreous and retina in newborn (1-3- day-old), young adult (2-month-old), and aging (12-month-old) rats, as well as their role as regulators of vitreous components. MicroRNAs are small, non-coding RNAs that post-transcriptionally regulate gene expression. Our results showed detection of all investigated let-7 isoforms (let-7a, let-7b, let-7c, let-7d, let-7e, let-7f and let-7i) in the retina and vitreous. Although most let-7 members were significantly upregulated in the vitreous during development, only let-7b, let-7c, and let-7e followed this same expression pattern in the retina. Let-7b and -7c increased in aging vitreous as well, and were expressed in vitro by Müller glial cells and their extracellular vesicles. Moreover, let-7 targeted hyaluronan synthase 2 (Has2) mRNA, a synthesizing enzyme of hyaluronan. These observations indicate that let-7 function is important during retina and vitreous development, and that isoforms of let-7 increased with aging, potentially modulating hyaluronan content.

Development of the visual system of anchovy larvae, Engraulis anchoita: A microanatomical description.

During the early ontogeny of fish larvae, the accurate development of the visual system plays a key role, because it is involved in locating food, orientation, selection of favorable habitat, and evasion of predators. The structure of the eye of the fish is typical of vertebrates, with some modifications related to the aquatic environment. In the present work, we describe the development of the larval eye of Engraulis anchoita for the first time. Larvae were collected at the Permanent Station of Environmental Studies (EPEA) in coastal waters of the Southwestern Atlantic Ocean during research cruises in 2015 and 2016. We describe the histology of the retina layers, determine the beginning of the functionality of the eye, and discuss a possible synchronization with the development of the digestive tract. This study provides information about the biology of E. anchoita, the most abundant fish species in the southwestern Atlantic Ocean. Also, recent studies have shown responses of the retina and other tissues to the increase in environmental acidity. Therefore, results of this study are also discussed with respect to the possible effect of acidification on the larvae of this species. The continuity of the time series developed at the EPEA will allow monitoring the effect of long-term environmental and biological variables on the early ontogeny of anchovy in the context of climate change. The high commercial fishing potential of E. anchoita due to its high abundance, as well as its essential role in the trophic web of other commercially valuable fishing resources of Argentina, reinforce the need to continue deepening knowledge about this species. Research highlights: Eyes of Engraulis anchoita larvae are functional from early larval stages. At hatching, the retina is formed by only few layers from which the other layers differentiates during ontogeny. Focal distance increases with larval growth.

Retinal morphology in Astyanax mexicanus during eye degeneration.

The teleost Astyanax mexicanus is one species extant in two readily available forms. One that lives in Mexican rivers and various convergent forms that live in nearby caves. These fish are born with eyes but in the cavefish, they degenerate during development. It is known that the lens of cavefish undergoes apoptosis and that some cells in the neuroretina also die. It has not been described, however, if glia and various components of the neuroretina form before complete eye degeneration. Here we examined the development of the retina of the closest living ancestor that lives in the rivers and two independently adapted of cavefish. We report that although the neuroretina is smaller and more compact, it has all cell types and layers including amacrine cells and Müller glia. While various makers for photoreceptors are present in the cavefish inner segments, the outer segments of the photoreceptors in cavefish are missing from the earliest stages examined. This shows that the machinery for visual transducing discs might still be present but not organized in one part of the cell. It is interesting to note that the deficiencies in Astyanax cavefish resemble retinal diseases, such as retinitis pigmentosa.

Small Molecule GSK-J1 Affects Differentiation of Specific Neuronal Subtypes in Developing Rat Retina.

Histone post-translational modification has been shown to play a pivotal role in regulating gene expression and fate determination during the development of the central nervous system. Application of pharmacological blockers that control histone methylation status has been considered a promising avenue to control abnormal developmental processes and diseases as well. In this study, we focused on the role of potent histone demethylase inhibitor GSK-J1 as a blocker of Jumonji domain-containing protein 3 (Jmjd3) in early postnatal retinal development. Jmjd3 participates in different processes such as cell proliferation, apoptosis, differentiation, senescence, and cell reprogramming via demethylation of histone 3 lysine 27 trimethylation status (H3K27 me3). As a first approach, we determined the localization of Jmjd3 in neonate and adult rat retina. We observed that Jmjd3 accumulation is higher in the adult retina, which is consistent with the localization in the differentiated neurons, including ganglion cells in the retina of neonate rats. At this developmental age, we also observed the presence of Jmjd3 in undifferentiated cells. Also, we confirmed that GSK-J1 caused the increase in the H3k27 me3 levels in the retinas of neonate rats. We next examined the functional consequences of GSK-J1 treatment on retinal development. Interestingly, injection of GSK-J1 simultaneously increased the number of proliferative and apoptotic cells. Furthermore, an increased number of immature cells were detected in the outer plexiform layer, with longer neuronal processes. Finally, the influence of GSK-J1 on postnatal retinal cytogenesis was examined. Interestingly, GSK-J1 specifically caused a significant decrease in the number of PKCα-positive cells, which is a reliable marker of rod-on bipolar cells, showing no significant effects on the differentiation of other retinal subtypes. To our knowledge, these data provide the first evidence that in vivo pharmacological blocking of histone demethylase by GSK-J1 affects differentiation of specific neuronal subtypes. In summary, our results indisputably revealed that the application of GSK-J1 could influence cell proliferation, maturation, apoptosis induction, and specific cell determination. With this, we were able to provide evidence that this small molecule can be explored in therapeutic strategies for the abnormal development and diseases of the central nervous system.

Flaxseed used since pregnancy by the mother and after weaning by the offspring benefits the retina and optic nerve development in rats.

OBJECTIVE: This study aimed to evaluate the influence of a diet based on flaxseed upon the development of the nervous system, more specifically, the optic nerve and retina. METHODS: Rats were divided into three groups: Control (CG), Flaxseed (FG), and Modified Control (MCG). The analyses were performed in the offspring (n = 6/group) at the immediate postnatal period (P0), 14 d of life (P14) and 30 d of life (P30). Descriptive analysis and histomorphometry of optic nerve and retina were performed. RESULTS: There was a great evolution in the development of the nervous fascicles, connective trabeculae, and blood vessels, when comparing the three ages studied, and these characteristics were more evident in FG at all three ages. The P0, P14, and P30 retina showed similar morphology to that described in the literature. In histomorphometry, at P14, the FG presented the retina and its layers with significant increase in thickness, except for internal granular and ganglionar, whereas MCG had greater retina and photoreceptor layers thickness, inner plexiform and external granular when compared with CG (p < .05). CONCLUSION: The use of flaxseed in the pre-and postnatal period displays favourable influence on the development of rat optic nerve and retina, probably leading to myelination.

Analysis of melanopsin gene expression in the rabbit retina at different ages.

Melanopsin is the photopigment of intrinsically photosensitive retinal ganglion cells that mediate non-visual responses to light. The aim of this study was to describe and analyze melanopsin gene expression in the rabbit retina at different ages and compare its expression with the prototypic gene of retinal ganglion cells (Thy-1 gene). Expression levels of OPN4, Thy-1, and GADPH genes were measured by real-time PCR at 3, 4, 8, 11, 12, 17, 19, 20, 23, 27, 32, and 47 postnatal days. We also regrouped the days before and after day 12 of life (pre-photic and post-photic stage, respectively). Average expression of the OPN4 gene between days was similar (P = 0.713), but was statistically different in the Thy-1 gene (P = 0.004). Also, no significant differences were found in OPN4 gene expression pre-photic and post-photic stage (P = 0.629); however, Thy-1 expression was higher in the pre-photic stage, almost double, than in the post-photic stage, with significant differences (P = 0.001). This is the first report describing OPN4 gene expression in the rabbit retina at different ages. We demonstrated that the OPN4 gene is constantly expressed at all early stages, even before the onset of photoentrainment by the pups and that Thy-1 and OPN4 gene expressions are out of phase.

Caffeine exposure alters adenosine system and neurochemical markers during retinal development.

Evidence points to beneficial properties of caffeine in the adult central nervous system, but teratogenic effects have also been reported. Caffeine exerts most of its effects by antagonizing adenosine receptors, especially A1 and A2A subtypes. In this study, we evaluated the role of caffeine on the expression of components of the adenosinergic system in the developing avian retina and the impact of caffeine exposure upon specific markers for classical neurotransmitter systems. Caffeine exposure (5-30 mg/kg by in ovo injection) to 14-day-old chick embryos increased the expression of A1 receptors and concomitantly decreased A2A adenosine receptors expression after 48 h. Accordingly, caffeine (30 mg/kg) increased [(3) H]-8-cyclopentyl-1,3-dipropylxanthine (A1 antagonist) binding and reduced [(3) H]-ZM241385 (A2A antagonist) binding. The caffeine time-response curve demonstrated a reduction in A1 receptors 6 h after injection, but an increase after 18 and 24 h. In contrast, caffeine exposure increased the expression of A2A receptors from 18 and 24 h. Kinetic assays of [(3) H]-S-(4-nitrobenzyl)-6-thioinosine binding to the equilibrative adenosine transporter ENT1 revealed an increase in Bmax with no changes in Kd , an effect accompanied by an increase in adenosine uptake. Immunohistochemical analysis showed a decrease in retinal content of tyrosine hydroxylase, calbindin and choline acetyltransferase, but not Brn3a, after 48 h of caffeine injection. Furthermore, retinas exposed to caffeine had increased levels of phosphorylated extracellular signal-regulated kinase and cAMP-response element binding protein. Overall, we show an in vivo regulation of the adenosine system, extracellular signal-regulated kinase and cAMP-response element binding protein function and protein expression of specific neurotransmitter systems by caffeine in the developing retina. The beneficial or maleficent effects of caffeine have been demonstrated by the work of different studies. It is known that during animal development, caffeine can exert harmful effects, impairing the correct formation of CNS structures. In this study, we demonstrated cellular and tissue effects of caffeine's administration on developing chick embryo retinas. Those effects include modulation of adenosine receptors (A1 , A2 ) content, increasing in cAMP response element-binding protein (pCREB) and extracellular signal-regulated kinase phosphorylation (pERK), augment of adenosine equilibrative transporter content/activity, and a reduction of some specific cell subpopulations. ENT1, Equilibrative nucleoside transporter 1.

Intravitreous Injection of Interleukin-6 Leads to a Sprouting in the Retinotectal Pathway at Different Stages of Development.

OBJECTIVE: The development of retinotectal pathways form precise topographical maps is usually completed by the third postnatal week. Cytokines participate in the development and plasticity of the nervous system. We have previously shown that in vivo treatment with interleukin 2 disrupts the retinocollicular topographical order in early stages of development. Therefore, we decided to study the effect of a single intravitreous injection of IL-6 upon retinotectal circuitry in neonates and juvenile rats. MATERIALS AND METHODS: Lister Hooded rats received an intravitreous injection of IL-6 (50 ng/ml) or vehicle (PBS) at either postnatal day (PND)10 or PND30 and the ipsilateral retinotectal pathway was evaluated 4 or 8 days later, respectively. RESULTS: Our data showed that, at different stages of development, a single IL-6 intravitreous treatment did not produce an inflammatory response and increased retinal axon innervation throughout the visual layers of the superior colliculus. CONCLUSIONS: Taken together, our data provide the first evidence that a single intravitreous injection with IL-6 leads to sprouting in the subcortical visual connections and suggest that small changes in IL-6 levels might be sufficient to impair the correct neuronal circuitry fine-tuning during brain development.

Nicotine-induced plasticity in the retinocollicular pathway: Evidence for involvement of amyloid precursor protein.

During early postnatal development retinocollicular projections undergo activity-dependent synaptic refinement that results in the formation of precise topographical maps in the visual layers of the superior colliculus (SC). Amyloid Precursor Protein (APP) is a widely expressed transmembrane glycoprotein involved in the regulation of several aspects of neural development, such as neurite outgrowth, synapse formation and plasticity. Stimulation of cholinergic system has been found to alter the expression and processing of APP in different cell lines. Herein, we investigated the effect of nicotine on the development of retinocollicular pathway and on APP metabolism in the SC of pigmented rats. Animals were submitted to intracranial Elvax implants loaded with nicotine or phosphate-buffered saline (vehicle) at postnatal day (PND) 7. The ipsilateral retinocollicular pathway of control and experimental groups was anterogradely labeled either 1 or 3 weeks after surgery (PND 14 or PND 28). Local nicotine exposure produces a transitory sprouting of uncrossed retinal axons outside their main terminal zones. Nicotine also increases APP content and its soluble neurotrophic fragment sAPPα. Furthermore, nicotine treatment upregulates nicotinic acetylcholine receptor α7 and ß2 subunits. Taken together, these data indicate that nicotine disrupts the ordering and topographic mapping of axons in the retinocollicular pathway and facilitates APP processing through the nonamyloidogenic pathway, suggesting that sAPPα may act as a trophic agent that mediates nicotine-induced morphological plasticity.

Estudio morfológico de la retina de salmones (Salmo salar) / Morphological study of the retina of salmon (Salmo salar)

La retina de peces teleósteos como pez cebra, se ha transformado en un importante modelo para el estudio de la plasticidad neuronal y la neurogénesis. Se ha demostrado además que la retina experimenta cambios ontogenéticos para adaptarse a distintos medios ambientes durante su vida. Este estudio tiene como objetivo describir el desarrollo ontogenético de la retina del alevín de salmón desde la eclosión hasta la fase de juvenil. Se trabajó con 30 salmones divididos en tres grupos de 10. Grupo I: recién eclosionados, con saco vitelino y 18 mm de longitud. Grupo II: sin saco vitelino y 30 mm de longitud. Grupo III: 100 mm de longitud. Cinco alevinesde cada grupo fueron procesados según el protocolo de Hanken & Wassersug para medir los diámetros dorsoventral y nasal-temporal utilizando el cartílago que protege al globo ocular. Los restantes cinco ejemplares fueron seccionados con micrótomo Microm en forma seriada (5 µm) y procesados con técnica H&E/Azul de Alcián. Se midieron las capas de la retina en un microscopio óptico Zeiss, con cámara Powershot incorporada y con un software Image Tool 3.0. El Grupo 1 presentó grandes ojos pigmentados, con aspecto de copa óptica embrionaria, la retina está estratificada en capas. La Capa Nuclear Interna (CNI) mide 62±10 µm y la capa plexiforme interna (CPI) 10±2 µm. El Grupo 2 presenta cambios en el espesor de ellas. La CNI disminuye su espesor a 45±8 µm y la Plexiforme aumenta a 25±5 µm. En los peces juveniles del Grupo 3, la CNI alcanza el espesor mínimo (15±3 µm), por el contrario, la capa Plexiforme interna aumenta su espesor hasta alcanzar (70±10 µm). En los tres grupos estudiados observamos en la periferia de la retina una zona proliferativa germinativa, que corresponde a un remanente del neuroepitelio embrionario, responsable del crecimiento continuado de la retina. La retina de los salmones puede ser también un importante modelo para el estudio de la ontogenia, la plasticidad neuronal y la neurogénesis. Esta neurogénesis en la retina de peces facilita la reordenación celular a lo largo de la ontogenia, lo que potencialmente permite la optimización del sistema visual a los cambios en las demandas visuales. Este estudio puede ser de utilidad para facilitar el diagnóstico en las patologías de ojo en salmonicultura y también puede contribuir a conocer mejor la regeneración de tejidos. Por otro lado, con estudios posteriores, la neurogénesis de la retina de peces podría extrapolarse al tratamiento de enfermedades humanas con daño a nivel retineal, tales como glaucoma, desprendimiento de retina y retinopatía diabética.

The retina of teleost fish zebrafish, has become an important model for studying neuronal plasticity and neurogenesis. It was further shown that the retina undergoes ontogenetic changes to adapt to different environments during their lifetime. This study aims to describe the ontogenetic development of the retina of juvenile salmon from hatching to the juvenile stage. We worked with 30 salmon divided into three groups of 10. Group I: newly hatched with yolk sac and 18 mm in length. Group II: without yolk sac and 30 mm in length. Group III: 100 mm long. Five fry each group were processed according to the protocol of Hanken & Wassersug to measure dorsoventral and nasal-temporal diameters using the cartilage that protects the eyeball. The remaining five specimens were sectioned with a microtome Microm serially (5 µm) and processed with technical H-E / Alcian blue. The layers of the retina were measured on a Zeiss optical microscope with camera Powershot built and with Image Tool 3.0 software. Group 1 showed large pigmented eyes, looking embryonic optic cup, the retina is stratified in layers. The inner nuclear layer (CNI) measured 62±10 microns and the inner plexiform layer (CPI) 10±2 µm. Group 2 presents changes in the thickness of them. The CNI decreases in thickness to 45±8 µm and the plexiform increased to 25±5 µm. In juvenile fish of group 3, the CNI reaches the minimum thickness (15±3 µm), by contrast, the inner plexiform layer thickness increases up to (70±10 µm). In the three groups observed in the periphery of the retina one proliferative germinative zone, which corresponds to a remnant of the embryonic neural epithelium responsible for the continued growth of the retina. The retina of the salmon can also be an important model for the study of ontogeny, neuronal plasticity and neurogenesis. This retinal neurogenesis fish rearrangement facilitates cell along ontogeny, potentially allowing optimization of the visual system to changes in the visual demands. This study may be useful to help diagnose pathologies in eye salmon and can also contribute to better understand tissue regeneration. On the other hand, with later studies, fish's retinal neurogenesis could be extrapolated to the treatment of human retinal diseases, such us glaucoma, retinal detachment o diabetic retinopathy.

A critical period for omega-3 nutritional supplementation in the development of the rodent visual system.

Retinocollicular connections form precise topographical maps that are normally completed through the selective elimination of misplaced axons and the stabilization of topographically ordered axon terminals during early development. Omega-3 fatty acids, acquired exclusively through the diet, and its main metabolite, docosahexaenoic acid (DHA), are involved in brain development and synaptic maturation. We have previously shown that the nutritional restriction of omega-3/DHA results in abnormal retinocollicular topographical fine-tuning. Therefore, we studied the role of omega-3 fatty acids nutritional supplementation and the developmental time windows during which this postnatal supplementation would restore normal topographical maps in the visual system. Female rats and their litters were chronically fed with either control (soy oil) or restricted omega-3 (coconut oil) diets. Fish oil supplementation was introduced between either postnatal day (PND) 7-13, PND7-28 or PND21-42. At PND13, PND28 or PND42, animals received an anterograde eye injection of a neuronal tracer to visualize retinocollicular axons. Confirming previous observations we found that an omega-3/DHA deficiency resulted in an abnormally high innervation density of retinal axons at the visual layers of the superior colliculus (SC). Although a short-term fish oil supplementation between PND7-13 could not restore normal retinocollicular topography, an extended treatment between PND7-28 completely recovered normal innervation densities of retinotectal axons. However, a late onset supplementation protocol, between PND28-42, was no longer effective in the restoration of the abnormal topographical pattern induced by an early omega-3 nutritional malnutrition. The results suggest a critical period for omega3/DHA dietary intake for the proper development of visual topographical maps.

Functional plasticity of GAT-3 in avian Müller cells is regulated by neurons via a glutamatergic input.

GABA (γ-amino butyric acid) is the major inhibitory transmitter in the central nervous system and its action is terminated by specific transporters (GAT), found in neurons and glial cells. We have previously described that GAT-3 is responsible for GABA uptake activity in cultured avian Müller cells and that it operates in a Na(+) and Cl(-) dependent manner. Here we show that glutamate decreases [(3)H] GABA uptake in purified cultured glial cells up to 50%, without causing cell death. This effect is mediated by ionotropic glutamatergic receptors. Glutamate inhibition on GABA uptake is not reverted by inhibitors of protein kinase C or modified by agents that modulate cyclic AMP/PKA. Biotinylation experiments demonstrate that this reduction in GABA uptake correlates with a decrease in GAT-3 plasma membrane levels. Interestingly, both GAT-1 and GAT-3 mRNA levels are also decreased by glutamate. Conditioned media (CM) prepared from retinal neurons could also decrease GABA influx, and glutamate receptor antagonists (MK-801 + CNQX) were able to prevent this effect. However, glutamate levels in CM were not different from those found in fresh media, indicating that a glutamatergic co-agonist or modulator could be regulating GABA uptake by Müller cells in this scenario. In the whole avian retina, GAT-3 is present from embryonic day 5 (E5) increasing up to the end of embryonic development and post-hatch period exclusively in neuronal layers. However, this pattern may change in pathological conditions, which drive GAT-3 expression in Müller cells. Our data suggest that in purified cultures and upon extensive neuronal lesion in vivo, shown as a Brn3a reduced neuronal cells and an GFAP increased gliosis, Müller glia may change its capacity to take up GABA due to GAT-3 up regulation and suggests a regulatory interplay mediated by glutamate between neurons and glial cells in this process.

Leishmania, Babesia and Ehrlichia in urban pet dogs: co-infection or cross-reaction in serological methods?

INTRODUCTION: The present study was designed to assess the occurrence of co-infection or cross-reaction in the serological techniques used for detecting the anti-Leishmania spp., -Babesia canis vogeli and -Ehrlichia canis antibodies in urban dogs from an area endemic to these parasites. METHODS: The serum samples from dogs were tested for the Babesia canis vogeli strain Belo Horizonte antigen and Ehrlichia canis strain São Paulo by immunofluorescence antibody test (IFAT) and by anti-Leishmania immunoglobulin G (IgG) antibody detection to assess Leishmania infection. We used the following four commercial kits for canine visceral leishmaniasis: ELISA, IFAT, Dual Path Platform (DPP) (Bio Manguinhos(r)/FIOCRUZ/MS) and a rK39 RDT (Kalazar Detect Canine Rapid Test; Inbios). RESULTS : Of 96 serum samples submitted to serological assays, 4 (4.2%) were positive for Leishmania as determined by ELISA; 12 (12.5%), by IFAT; 14 (14.6%) by rK39 RDT; and 20 (20.8%), by DPP. Antibodies against Ehrlichia and Babesia were detected in 23/96 (23.9%) and 30/96 (31.2%) samples, respectively. No significant association was identified between the results of tests for detecting Babesia or Ehrlichia and those for detecting Leishmania (p-value>0.05). CONCLUSIONS: In the present study, we demonstrated co-infection with Ehrlichia or Babesia and Leishmania in dogs from Minas Gerais (Brazil); we also found that the serological tests that were used did not cross-react. .

The (Na(+)/K (+))-ATPase activity in the developing rat retina: the role of insulin-like growth factor-I (IGF-I).

In this work, the (Na(+)/K(+))-ATPase activity was evaluated during the early stages of the postnatal development of rat retina and showed an almost three-time increase from P0 to P14. Expression of the three catalytic subunit isoforms (α1, α2, and α3) of the (Na(+)/K(+))-ATPase was also evaluated by immunoblot in the same period, but no correlation to the catalytic activity increment was observed. On the other hand, immunolocalization of these three α-catalytic isoforms in the developing retina showed an age-related pattern. Involvement of IGF-I in the stimulation of the (Na(+)/K(+))-ATPase was investigated. Our results demonstrate that the exogenous IGF-I (10 ng/mL) stimulates enzyme activity at the age of P7 only. Incubation of retinas with 10 µM I-OMe-AG 538 (inhibitor of the IGF-I receptor) indicates that the basal (Na(+)/K(+))-ATPase activity is sustained by endogenous IGF-I in P7 animals. These data were corroborated by an age-dependent decrease in the immunodetection of endogenous IGF-I as well as in the phosphorylation level of its cognate receptor in rat retina homogenates. The signaling pathway involved in IGF-I-induced modulation of the (Na(+)/K(+))-ATPase was also investigated. Our data show that the inhibitory effects induced by I-OMe-AG 538 and the PI 3-kinase inhibitor Ly 294002 on the basal (Na(+)/K(+))-ATPase activity were non-cumulative. Furthermore, IGF-I induced phosphorylation of PKB in a Ly 294002-sensitive manner. Together, these data demonstrate that the PI 3-kinase/PKB signaling pathway is involved in the IGF-I-sustained basal (Na(+)/K(+))-ATPase activity during the first 7 days of the postnatal development of rat retina.

Pleiotropic functions of pituitary adenylyl cyclase-activating polypeptide on retinal ontogenesis: involvement of KLF4 in the control of progenitor cell proliferation.

We showed previously that the neuropeptide pituitary adenylyl cyclase-activating polypeptide (PACAP) negatively regulates proliferation of postnatal rat retinal progenitor cells through the downregulation of cyclin D1 in a cAMP/protein kinase A dependent manner. In the present study, we describe by microarray analysis several putative PACAP targets regulated by different transcription factor families. One of these families is the Sp/Klf family of transcriptional factors capable of regulating cyclin D1, and among members, we demonstrate by immunocytochemistry that KLF4 is expressed throughout rat retinal development by retinal progenitor cells and in most differentiated cell types. Using retinal explants preparations, PACAP treatment can transiently increase Klf4 mRNA levels; from electrophoretic mobility shift assays, PACAP is also able to increase the nuclear KLF4 content. From these results, we suggest that KLF4 may be involved in the anti-proliferative effects of PACAP as one mechanism regulating progenitor cell transition from proliferation to differentiation throughout retinal development.

The nitric oxide-cGKII system relays death and survival signals during embryonic retinal development via AKT-induced CREB1 activation.

During early neurogenesis, retinal neuronal cells display a conserved differentiation program in vertebrates. Previous studies established that nitric oxide (NO) and cGMP accumulation regulate essential events in retinal physiology. Here we used pharmacological and genetic loss-of-function to investigate the effects of NO and its downstream signaling pathway in the survival of developing avian retinal neurons in vitro and in vivo. Six-day-old (E6) chick retinal cells displayed increased calcium influx and produced higher amounts of NO when compared with E8 cells. L-arginine (substrate for NO biosynthesis) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP; a nitrosothiol NO donor) promoted extensive cell death in E6 retinas, whereas in E8 both substances decreased apoptosis. The effect of NO at both periods was mediated by soluble guanylyl cyclase (sGC) and cGMP-dependent kinase (cGK) activation. In addition, shRNA-mediated cGKII knockdown prevented NO-induced cell death (E6) and cell survival (E8). This, NO-induced cell death or cell survival was not correlated with an early inhibition of retinal cell proliferation. E6 cells also responded differentially from E8 neurons regarding cyclic AMP-responsive element-binding protein (CREB) activation in the retina in vivo. NO strongly decreased nuclear phospho-CREB staining in E6 but it robustly enhanced CREB phosphorylation in the nuclei of E8 neurons, an effect that was completely abrogated by cGKII shRNAs at both embryonic stages. The ability of NO in regulating CREB differentially during retinal development relied on the capacity of cGKII in decreasing (E6) or increasing (E8) nuclear AKT (V-Akt murine thymoma viral oncogene) activation. Accordingly, inhibiting AKT prevented both cGKII shRNA-mediated CREB upregulation in E6 and SNAP-induced CREB activation in E8. Furthermore, shRNA-mediated in vivo cGKII or in vitro CREB1 knockdown confirmed that NO/cGKII dualistically regulated the downstream CREB1 pathway and caspase activation in the chick retina to modulate neuronal viability. These data demonstrate that NO-mediated cGKII signaling may function to control the viability of neuronal cells during early retinal development via AKT/CREB1 activity.

Developmental and functional expression of miRNA-stability related genes in the nervous system.

In the nervous system, control of gene expression by microRNAs (miRNAs) has been investigated in fundamental processes, such as development and adaptation to ambient demands. The action of these short nucleotide sequences on specific genes depends on intracellular concentration, which in turn reflects the balance of biosynthesis and degradation. Whereas mechanisms underlying miRNA biogenesis has been investigated in recent studies, little is known about miRNA-stability related proteins. We first detected two genes in the retina that have been associated to miRNA stability, XRN2 and PAPD4. These genes are highly expressed during retinal development, however with distinct subcellular localization. We investigated whether these proteins are regulated during specific phases of the cell cycle. Combined analyses of nuclei position in neuroblastic layer and labeling using anti-cyclin D1 revealed that both proteins do not accumulate in S or M phases of the cell cycle, being poorly expressed in progenitor cells. Indeed, XRN2 and PAPD4 were observed mainly after neuronal differentiation, since low expression was also observed in astrocytes, endothelial and microglial cells. XRN2 and PAPD4 are expressed in a wide variety of neurons, including horizontal, amacrine and ganglion cells. To evaluate the functional role of both genes, we carried out experiments addressed to the retinal adaptation in response to different ambient light conditions. PAPD4 is upregulated after 3 and 24 hours of dark- adaptation, revealing that accumulation of this protein is governed by ambient light levels. Indeed, the fast and functional regulation of PAPD4 was not related to changes in gene expression, disclosing that control of protein levels occurs by post-transcriptional mechanisms. Furthermore, we were able to quantify changes in PAPD4 in specific amacrine cells after dark -adaptation, suggesting for circuitry-related roles in visual perception. In summary, in this study we first described the ontogenesis and functional expression of these two miRNA-stability related proteins in the retina.

Calbindin-D28k and calretinin in chicken inner retina during postnatal development and neuroplasticity by dim red light.

Members of the family of calcium binding proteins (CBPs) are involved in the buffering of calcium (Ca2+) by regulating how Ca2+ can operate within synapses or more globally in the entire cytoplasm and they are present in a particular arrangement in all types of retinal neurons. Calbindin D28k and calretinin belong to the family of CBPs and they are mainly co-expressed with other CBPs. Calbindin D28k is expressed in doubles cones, bipolar cells and in a subpopulation of amacrine and ganglion neurons. Calretinin is present in horizontal cells as well as in a subpopulation of amacrine and ganglion neurons. Both proteins fill the soma at the inner nuclear layer and the neuronal projections at the inner plexiform layer. Moreover, calbindin D28k and calretinin have been associated with neuronal plasticity in the central nervous system. During pre and early postnatal visual development, the visual system shows high responsiveness to environmental influences. In this work we observed modifications in the pattern of stratification of calbindin immunoreactive neurons, as well as in the total amount of calbindin through the early postnatal development. In order to test whether or not calbindin is involved in retinal plasticity we analyzed phosphorylated p38 MAPK expression, which showed a decrease in p-p38 MAPK, concomitant to the observed decrease of calbindin D28k. Results showed in this study suggest that calbindin is a molecule related with neuroplasticity, and we suggest that calbindin D28k has significant roles in neuroplastic changes in the retina, when retinas are stimulated with different light conditions.

ATP induces the death of developing avian retinal neurons in culture via activation of P2X7 and glutamate receptors.

Previous data suggest that nucleotides are important mitogens in the developing retina. Here, the effect of ATP on the death of cultured chick embryo retina cells was investigated. In cultures obtained from retinas of 7-day-old chick embryos (E7) that were cultivated for 2 days (E7C2), both ATP and BzATP induced a ∼30 % decrease in cell viability that was time- and dose-dependent and that could be blocked by 0.2 mM oxidized ATP or 0.3 µM KN-62. An increase in cleaved caspase-3 levels and in the number of TUNEL-positive cells was observed when cultures were incubated with 3 mM ATP and immunolabeling for cleaved-caspase 3 was observed over neurons but not over glial cells. ATP-dependent cell death was developmentally regulated, the maximal levels being detected by E7C2-3. Nucleotides were able to increase neuronal ethidium bromide and sulforhodamine B uptake in mixed and purified neuronal cultures, an effect that was blocked by the antagonists Brilliant Blue G and oxidized ATP. In contrast, nucleotide-induced cell death was observed only in mixed cultures, but not in purified cultures of neurons or glia. ATP-induced neuronal death was blocked by the glutamatergic antagonists MK801 and DNQX and activation of P2X7 receptors by ATP decreased the uptake of [(3)H]-D-aspartate by cultured glial cells with a concomitant accumulation of it in the extracellular medium. These results suggest that ATP induces apoptosis of chick embryo retinal neurons in culture through activation of P2X7 and glutamate ionotropic receptors. Involvement of a P2X7 receptor-mediated inhibition of the glial uptake of glutamate is suggested.