Melatonin and the time window for the expression of the alpha 8 nicotinic acetylcholine receptor in the membrane of chick retinal cells in culture

We have previously shown that melatonin influences the development of alpha 8 nicotinic acetylcholine receptor (nAChR) by measurement of the acetylcholine-induced increase in the extracellular acidification rate (ECAR) in chick retinal cell cultures. Cellular differentiation that takes place between DIV (days in vitro) 4 and DIV 5 yields cells expressing alpha 8 nAChR and results in a significant increase in the ECAR acetylcholine-induced. Blocking melatonin receptors with luzindole for 48 h suppresses the development of functional alpha 8 nAChR. Here we investigated the time window for the effect of melatonin on retinal cell development in culture, and whether this effect was dependent on an increase in the expression of alpha 8 nAChR. First, we confirmed that luzindole was inhibiting the effects of endogenous melatonin, since it increases 2-[(125)I] iodomelatonin (23 pM) binding sites density in a time-dependent manner. Then we observed that acute (15, 60 min, or 12 h) luzindole treatment did not impair acetylcholine-induced increase in the ECAR mediated by activation of alpha 8 nAChR at DIV 5, while chronic treatment (from DIV 3 or DIV 4 till DIV 5, or DIV 3.5 till DIV 4.5) led to a time-dependent reduction of the increase in the acetylcholine-induced ECAR. The binding parameters for [(125)I]-alpha-bungarotoxin (10 nM) sites in membrane were unaffected by melatonin suppression that started at DIV 3. Thus, melatonin surges in the time window that occurs at the final stages of chick retinal cell differentiation in culture is essential for development of the cells expressing alpha 8 nAChR subtype in full functional form. (C) 2010 ISDN. Published by Elsevier Ltd. All rights reserved.

TRPV1 receptors modulate retinal development

We investigated the possible participation of TRPV1 channels in retinal apoptosis and overall development. Retinas from newborn, male albino rats were treated in vitro with capsazepine, a TRPV1 antagonist. The expression of cell cycle markers was not changed after TRPV1 blockade, whereas capsazepine reduced the number of apoptotic cells throughout the retina,increased ERK1/2 and p38 phosphorylation and slightly reduced JNK phosphorylation. The expression of BAD, Bcl-2, as well as integral and cleaved capsase-3 were similar in all experimental conditions. Newborn rats were kept for 2 months after receiving high doses of capsazepine. In their retinas, calbindin and parvalbumin protein levels were upregulated, but only the number of amacrine-like, parvalbumin-positive cells was increased. The numbers of calretinin, calbindin, ChAT, vimentin, PKC-alpha and GABA-positive cells were similar in both conditions. Protein expression of synapsin Ib was also increased in the retinas of capsazepine-treated rats. Calretinin, vimentin, GFAP, synapsin Ia, synaptophysin and light neurofilament protein levels were not changed when compared to control values. Our results indicate that TRPV1 channels play a role in the control of the early apoptosis that occur during retinal development, which might be dependent on MAPK signaling. Moreover, it seems that TRPV1 function might be important for neuronal and synaptic maturation in the retina. (C) 2011 ISDN. Published by Elsevier Ltd. All rights reserved.

Pharmacological properties of purinergic receptors and their effects on proliferation and induction of neuronal differentiation of P19 embryonal carcinoma cells

We have used P19 embryonal carcinoma cells as in vitro model for early neurogenesis to study ionotropic P2X and metabotropic P2Y receptor-induced Ca2+ transients and their participation in induction of proliferation and differentiation. In embryonic P19 cells, P2Y(1), P2Y(2) and P2X(4) receptors or P2X-heteromultimers with similar P2X4 pharmacology were responsible for ATP and ATP analogue-induced Ca2+ transients. In neuronal-differentiated cells, P2Y(2), P2Y(6), P2X(2) and possibly P2X(2)/P2X(6) heteromeric receptors were the major mediators of the elevations in intracellular free calcium concentration [Ca2+](i). We have collected evidence for the involvement of metabotropic purinergic receptors in proliferation induction of undifferentiated and neural progenitor cells by using a BrdU-incorporation assay. ATP-, UTP-, ADP-, 2-MeS-ATP- and ADP-beta S-induced proliferation in P19 cells was mediated by P2Y, and P2Y2 receptors as judged from pharmacological profiles of receptor responses. ATP-provoked acceleration of neuronal differentiation, determined by analysis of nestin and neuron-specific enolase gene and protein expression, also resulted from P2Y, and P2Y2 receptor activation. Proliferation- and differentiation-induction involved the activation of inositol-trisphosphate sensitive intracellular Ca2+ stores. (C) 2008 ISDN. Published by Elsevier Ltd. All rights reserved.

Connexin-mediated communication controls cell proliferation and is essential in retinal histogenesis

Connexin (Cx) channels and hemichannels are involved in essential processes during nervous system development such as apoptosis, propagation of spontaneous activity and interkinetic nuclear movement. In the first part of this study, we extensively characterized Cx gene and protein expression during retinal histogenesis. We observed distinct spatio-temporal patterns among Studied Cx and an overriding, ubiquitous presence of Cx45 in progenitor cells. The role of Cx-mediated communication was assessed by using broad-spectrum (carbenoxotone, CBX) and Cx36/Cx50 channel-specific (quinine) blockers. In vivo application of CBX, but not quinine, caused remarkable reduction in retinal thickness, suggesting changes in cell proliferation/apoptosis ratio. Indeed, we observed a decreased number of mitotic cells in CBX-injected retinas, with no significant changes in the expression of PCNA, a marker for cells in proliferative state. Taken together, Our results pointed a pivotal role of Cx45 in the developing retina. Moreover, this study revealed that Cx-mediated Communication is essential in retinal histogenesis, particularly in the control of cell proliferation. (C) 2009 ISDN. Published by Elsevier Ltd. All rights reserved.

Ontogenetic expression of the vanilloid receptors TRPV1 and TRPV2 in the rat retina

The present study aimed to analyze the gene and protein expression and the pattern of distribution of the vanilloid receptors TRPV1 and TRPV2 in the developing rat retina. During the early phases of development, TRPV1 was found mainly in the neuroblastic layer of the retina and in the pigmented epithelium. In the adult, TRPV1 was found in microglial cells, blood vessels, astrocytes and in neuronal structures, namely synaptic boutons of both retina] plexiform layers, as well as in cell bodies of the inner nuclear layer and the ganglion cell layer. The pattern of distribution of TRPV1 was mainly punctate, and there was higher TRPV1 labeling in the peripheral retina than in central regions. TRPV2 expression was quite distinct. its expression was virtually undetectable by immunoblotting before P1, and that receptor was found by immunohistochemistry only by postnatal day 15 (PI 5). RNA and protein analysis showed that the adult levels are only reached by P60, which includes small processes in the retinal plexiform layers, and labeled cellular bodies in the inner nuclear layer and the ganglion cell layer. There was no overlapping between the signal observed for both receptors. in conclusion, our results showed that the patterns of distribution of TRPV1 and TRPV2 are different during the development of the rat retina, suggesting that they have specific roles in both visual processing and in providing specific cues to neural development. (C) 2009 ISDN. Published by Elsevier Ltd. All rights reserved.

Homocysteine induces oxidative stress, inflammatory infiltration, fibrosis and reduces glycogen/glycoprotein content in liver of rats

Hyperhomocysteinemia has been related to various diseases, including homocystinuria, neurodegenerative and hepatic diseases. In the present study we initially investigated the effect of chronic homocysteine administration on some parameters of oxidative stress, named total radical-trapping antioxidant potential, total antioxidant reactivity, catalase activity, chemiluminescence, thiobarbituric acid-reactive substances, and total thiol content in liver of rats. We also performed histological analysis, evaluating steatosis, inflammatory infiltration, fibrosis, and glycogen/glycoprotein content in liver tissue sections from hyperhomocysteinemic rats. Finally, we evaluated the activities of aminotransferases in liver and plasma of hyperhomocysteinemic rats. Wistar rats received daily subcutaneous injection of Hcy from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, liver and plasma were collected. Hyperhomocysteinemia decreased antioxidant defenses and total thiol content, and increased lipid peroxidation in liver of rats, characterizing a reliable oxidative stress. Histological analysis indicated the presence of inflammatory infiltrate, fibrosis and reduced content of glycogen/glycoprotein in liver tissue sections from hyperhomocysteinemic rats. Aminotransferases activities were not altered by homocysteine. Our data showed a consistent profile of liver injury elicited by homocysteine, which could contribute to explain, at least in part, the mechanisms involved in human liver diseases associated to hyperhomocysteinemia. (C) 2009 ISDN. Published by Elsevier Ltd. All rights reserved.

A retrospective model of oocyte competence: global mRNA and housekeeping transcripts are not associated with in vitro developmental outcome

Oocyte developmental competence depends on maternal stores that support development throughout a transcriptionally silent period during early embryogenesis. Previous attempts to investigate transcripts associated with oocyte competence have relied on prospective models, which are mostly based on morphological. criteria. Using a retrospective model, we quantitatively compared mRNA among oocytes with different embryo development competence. A cytoplasm biopsy was removed from in vitro matured oocytes to perform comparative analysis of amounts of global polyadenylated (polyA) mRNA and housekeeping gene transcripts. After parthenogenetic activation of biopsied oocytes, presumptive zygotes were cultured individually in vitro and oocytes were classified according to embryo development: (i) blocked before the 8-cell stage; (ii) blocked between the 8-cell and morulae stages; or (iii) developed to the blastocyst stage. Sham-manipulated controls confirmed that biopsies did not alter development outcome. Total polyA mRNA amounts correlate with oocyte diameter but not with the ability to develop to the 8-cell and blastocyst stages. The last was also confirmed by relative quantification of GAPDH, H2A and Hprt1 transcripts. In conclusion, we describe a novel retrospective model to identify putative markers of development competence in single oocytes and demonstrate that global mRNA amounts at the metaphase II stage do not correlate with embryo development in vitro.

Cytoplasmic maturation of bovine oocytes: Structural and biochemical modifications and acquisition of developmental competence

Oocyte maturation is a long process during which oocytes acquire their intrinsic ability to support the subsequent stages of development in a stepwise manner, ultimately reaching activation of the embryonic genome. This process involves complex and distinct, although linked, events of nuclear and cytoplasmic maturation. Nuclear maturation mainly involves chromosomal segregation, whereas cytoplasmic maturation involves organelle reorganization and storage of mRNAs, proteins and transcription factors that act in the overall maturation process, fertilization and early embryogenesis. Thus, for didactic purposes, we subdivided cytoplasmic maturation into: (1) organelle redistribution, (2) cytoskeleton dynamics, and (3) molecular maturation. Ultrastructural analysis has shown that mitochondria, ribosomes, endoplasmic reticulum, cortical granules and the Golgi complex assume different positions during the transition from the germinal vesicle stage to metaphase II. The cytoskeletal microfilaments and microtubules present in the cytoplasm promote these movements and act on chromosome segregation. Molecular maturation consists of transcription, storage and processing of maternal mRNA, which is stored in a stable, inactive form until translational recruitment. Polyadenylation is the main mechanism that initiates protein translation and consists of the addition of adenosine residues to the 3` terminal portion of mRNA. Cell cycle regulators, proteins, cytoplasmic maturation markers and components of the enzymatic antioxidant system are mainly transcribed during this stage. Thus, the objective of this review is to focus on the cytoplasmic maturation process by analyzing the modifications in this compartment during the acquisition of meiotic competence for development. (c) 2009 Elsevier Inc. All rights reserved.

Consensus Statement: Chromosomal Microarray Is a First-Tier Clinical Diagnostic Test for Individuals with Developmental Disabilities or Congenital Anomalies

Chromosomal microarray (CMA) is increasingly utilized for genetic testing of individuals with unexplained developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD), or multiple congenital anomalies (MCA). Performing CMA and G-banded karyotyping on every patient substantially increases the total cost of genetic testing. The International Standard Cytogenomic Array (ISCA) Consortium held two international workshops and conducted a literature review of 33 studies, including 21,698 patients tested by CMA. We provide an evidence-based summary of clinical cytogenetic testing comparing CMA to G-banded karyotyping with respect to technical advantages and limitations, diagnostic yield for various types of chromosomal aberrations, and issues that affect test interpretation. CMA offers a much higher diagnostic yield (15%-20%) for genetic testing of individuals with unexplained DD/ID, ASD, or MCA than a G-banded karyotype (similar to 3%, excluding Down syndrome and other recognizable chromosomal syndromes), primarily because of its higher sensitivity for submicroscopic deletions and duplications. Truly balanced rearrangements and low-level mosaicism are generally not detectable by arrays, but these are relatively infrequent causes of abnormal phenotypes in this population (<1%). Available evidence strongly supports the use of CMA in place of G-banded karyotyping as the first-tier cytogenetic diagnostic test for patients with DD/ID, ASD, or MCA. G-banded karyotype analysis should be reserved for patients with obvious chromosomal syndromes (e.g., Down syndrome), a family history of chromosomal rearrangement, or a history of multiple miscarriages.

Expression Profile and Distribution of Efhc1 Gene Transcript During Rodent Brain Development

One of the putative causative genes for juvenile myoclonic epilepsy (JME) is EFHC1. We report here the expression profile and distribution of Efhc1 messenger RNA (mRNA) during mouse and rat brain development. Real-time polymerase chain reaction revealed that there is no difference in the expression of Efhc1 mRNA between right and left hemispheres in both species. In addition, the highest levels of Efhc1 mRNA were found at intra-uterine stages in mouse and in adulthood in rat. In common, there was a progressive decrease in Efhc1 expression from 1-day-old neonates to 14-day-old animals in both species. In situ hybridization studies showed that rat and mouse Efhc1 mRNAs are expressed in ependymal cells of ventricle walls. Our findings suggest that Efhc1 expression is more important during initial phases of brain development and that at this stage it could be involved in key developmental mechanisms underlying JME.

VSImG: A high frame rate bitmap based display system for neuroscience research

This paper describes a visual stimulus generator (VSImG) capable of displaying a gray-scale, 256 x 256 x 8 bitmap image with a frame rate of 500 Hz using a boustrophedonic scanning technique. It is designed for experiments with motion-sensitive neurons of the fly`s visual system, where the flicker fusion frequency of the photoreceptors can reach up to 500 Hz. Devices with such a high frame rate are not commercially available, but are required, if sensory systems with high flicker fusion frequency are to be studied. The implemented hardware approach gives us complete real-time control of the displacement sequence and provides all the signals needed to drive an electrostatic deflection display. With the use of analog signals, very small high-resolution displacements, not limited by the image`s pixel size can be obtained. Very slow image displacements with visually imperceptible steps can also be generated. This can be of interest for other vision research experiments. Two different stimulus files can be used simultaneously, allowing the system to generate X-Y displacements on one display or independent movements on two displays as long as they share the same bitmap image. (C) 2011 Elsevier B.V. All rights reserved.