Vladimir Flores

Actin filaments are associated with the septate junctions of invertebrates

Septate junctions are almost ubiquitous in the tissues of invertebrates but are never found in those of vertebrates. In spite of their widespread occurrence and hence obvious importance to the invertebrates, their precise function has remained elusive although they have been variously considered to be regions of cell-cell coupling, permeability barriers or adhesion sites. This report demonstrates that elements of the cytoskeletal system insert into the cytoplasmic face of septate junctions. Actin filaments, identified by virtue of their capacity to bind the S1 subfragment of rabbit myosin, are associated with the membranes of septate junctions. Cytochalasin D, an actin depolymerizer, leads to disorganization of the intramembrane components of these junctions. These data suggest that a primary role of septate junctions could be to maintain intercellular cohesion and hence tissue integrity. The assembly and localization of these junctions may be mediated, directly or indirectly, by the cytoplasmic actin filaments associated with their lateral membranes.

Development of GABA binding sites in chick embryo optic lobe: effect of Triton X-100

The temporal course of the development of GABA receptor sites in chick optic lohe was studied as a parameter of neuronal differentiation in the central nervous system. At 10 days of incubation, specific [3H]GABA binding was of 0.08 pmol/optic lobe and increased 7–8 fold between 12 and 16 days of incubation, reaching at 16 days a value of 0.60 pmol/optic lobe. This coincides with the period of arrival of the retinal fibers to the optic lobe. After this stage, the number of GABA binding sites decreased to a value of 0.35 pmol/optic lobe at hatching. After hatching a new increase appeared which reached at 5 days post‐hatching a value of 0.87 pmol/optic lobe. Scatchard analysis of the saturation binding data obtained at 16 days of incubation and at hatching revealed the presence of two binding sites: one with high affinity and the other with low affinity, while at 12 days of incubation, the earliest stage examined, only the low‐affinity binding site appeared. The high‐affinity binding site for [3H]GABA was inhibited by muscimol, GABA, and bicuculline (ic50: 0.006, 0.002 and 10 μM, respectively). These values correspond to the potencies shown by those compounds in the binding to the synaptic GABA receptor. Treatment of the synaptic membranes with Triton X‐100) showed a marked increase in the amount of specific [3H]GABA binding after 16 days of incubation reaching a 3‐fold increase at hatching. These results suggest that endogenous inhibitors of the higher affinity binding site, probably appear during this period.

Effects of light- and dark-rearing on the postnatal development of GABA receptor sites in the chick optic lobe

To investigate the ability of GABA receptor sites to undergo environmental‐dependent plastic changes, the postnatal developmental pattern of GABA receptors was studied under different levels of light stimulation, i.e. normal‐, light‐ and dark‐rearing. At hatching the specific binding of (3H)GABA was 1.74 ± 0.36 pmol/optic lobe. In normally reared chicks the number of GABA binding sites showed a transient increase with the highest value at the 6th day (7.0 ± 1.32 pmol/optic lobe). This value is higher than the one reached at the adult stage. Between the 3rd and 6th day, there was a 33.7% increase in specific [3H]GABA binding in light‐reared compared with normally reared animals (P < 0.05). In the dark‐reared chicks, the specific binding was 36.4% lower than that found in normally reared (P < 0.02). However, the changes in receptor density were transient since at the 17th day the number of GABA binding sites returned to adult levels. Scatchard analysis revealed that the differences observed in the high affinity GABA binding sites between the three groups were due to modifications in the total number of binding sites while the affinity remained unchanged. The maximal number of binding sites were: 2.71, 7.01 and 1.79 pmol/mg protein in normally, light‐ and dark‐reared chicks, respectively; while the apparent dissociation constants were unaffected: 3.2, 3.4 and 3.6 nM, respectively. These results show that, during postnatal development, different conditions of visual experience produce synaptic changes at the molecular level. These changes probably occur within a period of high plasticity, prior to the end of a critical period.

Effect of a simple visual pattern on the early postnatal development oF GABA Receptor sites in the chick optic lobe

It is known that manipulation of the visual environment results in changes in the developmental pattern of several neurotransmitter receptors and that the GABA receptor shows a high degree of plasticity in differential illumination experiments. In the present paper we investigated whether exposure to a visual pattern has a developmental effect on GABA receptor expression during early postnatal life. Two groups of newly hatched chicks were used: one was exposed to a simple and specific visual pattern and the other was deprived of any visual pattern. GABA receptors at each developmental stage were determined by binding experiments performed in a crude membrane fraction. Saturation studies were carried out in a fraction enriched in synaptic membranes. The developmental pattern of both high and low affinity GABA binding sites was affected by the visual pattern. This effect displays its maximal expression by the end of the first postnatal week. The modification in receptor expression was due to a change in the receptor density while the affinity was not affected.

uPA-uPAR Molecular Complex is Involved in Cell Signaling During Neuronal Migration and Neuritogenesis

Background: In the development of the central nervous system (CNS), neuronal migration and neuritogenesis are crucial processes for establishing functional neural circuits. This relies on the regulation exerted by several signaling molecules, which play important roles in axonal growth and guidance. The urokinase‐type plasminogen activator (uPA)—in association with its receptor—triggers extracellular matrix proteolysis and other cellular processes through the activation of intracellular signaling pathways. Even though the uPA‐uPAR complex is well characterized in nonneuronal systems, little is known about its signaling role during CNS development. Results: In response to uPA, neuronal migration and neuritogenesis are promoted in a dose‐dependent manner. After stimulation, uPAR interacts with α5‐ and β1‐integrin subunits, which may constitute an αβ‐heterodimer that acts as a uPA‐uPAR coreceptor favoring the activation of multiple kinases. This interaction may be responsible for the uPA‐promoted phosphorylation of focal adhesion kinase (FAK) and its relocation toward growth cones, triggering cytoskeletal reorganization which, in turn, induces morphological changes related to neuronal migration and neuritogenesis. Conclusions: uPA has a key role during CNS development. In association with its receptor, it orchestrates both proteolytic and nonproteolytic events that govern the proper formation of neural networks. Developmental Dynamics 243:676–689, 2014.

Developmental pattern of NADPH‐diaphorase positive neurons in chick optic tectum is sensitive to changes in visual stimulation

The chick retinotectal system is a suitable model to investigate the mechanisms involved in the establishment of synaptic connections in whose refinement nitric oxide was implicated. The purpose of this work was to describe the developmental pattern of the nitric oxide synthase (NOS)‐positive neurons as well as to determine if it is sensitive to changes in visual stimulation. The NADPH‐diaphorase histochemical method was used to describe and quantify NOS neurons in normally stimulated and subnormally stimulated chickens. Nine types of NOS neurons were identified; seven of them express NOS until adulthood, while two of them show only a transient expression. The developmental pattern of NOS neurons follows the process of laminar segregation. It can be divided into three phases. The first includes the onset of NOS expression in periventricular neurons and the formation of a deep network of NOS fibers during early development. These neurons do not show any significant change in subnormally stimulated animals. The second phase includes the appearance of two transient NOS populations of bipolar neurons that occupy the intermediate layers during the optic fibers ingrowth. One of them significantly changes in subnormally stimulated chicks. The third phase occurs when the transitory expression of bipolar neurons decreases. It includes NOS expression in six neuronal populations that innervate the superficial retinorecipient layers. Most of these cells suffer plastic changes in subnormally stimulated chicks. The diversity of neuronal types with regard to their morphology, location, and sensitivity to visual stimulation strongly suggests that they serve different functions. J. Comp. Neurol. 494:1007–1030, 2006.

Proteases with plasminogen activator activity in hamster oviduct.

At present the physiological role of most oviductal proteins remains unknown. In this work, we present evidence that the oviductal secretion as well as the crude oviductal tissue‐extract show proteolytic‐like esterase and amidase activity. The proteolytic activity of the oviductal enzymes was higher in the oviducts of superovulated hamster females than in those of normal ones, indicating that gonadotrophic hormones would stimulate the synthesis and secretion of these enzymes. Some of their properties were analyzed in the 15,600‐g supernatant of both oviductal tissue extracts (OE) and oviductal fluid (OF). The enzymatic activity toward the synthetic substrates p‐tosyl‐l‐arginine methyl ester‐HCl (TAME) and α‐N‐benzoyl‐dl‐arginine‐p‐nitroanilide HCl (BAPNA) was activated by calcium ions, reached a maximum at pH 7.5, and was inhibited by soybean trypsin inhibitor (SBTI), N‐α‐p‐tosyl‐l‐lysine chloromethyl ketone HCl (TLCK), phenyl methyl sulfonyl fluoride (PMSF), and benzamidine. The OE glycoprotein fraction recognized by WGA‐Sepharose affinity columns (37% total proteins) showed proteolytic activity with properties similar to the OE and OF enzymes. The protease activity could be ascribed to a plasminogen activator (PA) detected in the Triton X‐100 treated tissue crude membrane fraction (Triton‐CMF) and in the oviductal secretion of the superovulated females. In the Triton‐CMF fraction, 100% of the proteolytic activity was plasminogen‐dependent. The use of amiloride, a selective urokinase‐type plasminogen activator (uPA) inhibitor, shows that 90% of this activity was due to a tissue‐type plasminogen activator (tPA) and 10% to uPA whereas in the uterus 100% of the activity was tPA. Only a small percentage of the OF proteolytic activity was plasminogen‐dependent, probably due to the presence of PA inhibitors in this medium. Mol. Reprod. Dev. 55:47–54, 2000.

Methods for removing endogenous factors from CNS membrane preparations: Differences in [3H]GABA binding parameters and developmental-related effects

The present report describes a systematic study comparing and combining methods currently used for the removal of endogenous factors known to affect the interaction of GABA with its receptor. The effects of these methods were analyzed by performing [3H]GABA binding studies, and by measuring the amount of residual GABA left in the different membrane preparations. The effectiveness of these methods were also applied to different developmental stages. The results show that: 1) an exhaustive buffer washing procedure is necessary to accurately measure the maximal binding capacity (Bmax) of the low-affinity GABA binding site, and 2) the use of more drastic methods, including freeze-thawing and Triton treatment allows a clear demonstration of receptor heterogeneity and a precise measurement of the Bmax of the high-affinity GABA binding site as well as increases the affinity of the low-affinity site. The analysis of the Bmax values obtained with these different procedure in relation to the values of GABA removal, strongly indicates that the exhaustive washing procedure removes some unknown endogenous substances required for Triton treatment to exhibit its maximal effectiveness. Finally, a detailed analysis of Kd and Bmax values obtained with these three methods in the developing nervous tissue shows the existence of significant differences with regard to their effectiveness in removing endogenous substances when applied in different developmental stages.

The chick optic tectum developmental stages. A dynamic table based on temporal‐ and spatial‐dependent histogenetic changes: A structural, morphometric and immunocytochemical analysis

Development is often described as temporal sequences of developmental stages (DSs). When tables of DS are defined exclusively in the time domain they cannot discriminate histogenetic differences between different positions along a spatial reference axis. We introduce a table of DSs for the developing chick optic tectum (OT) based on time‐ and space‐dependent changes in quantitative morphometric parameters, qualitative histogenetic features and immunocytochemical pattern of several developmentally active molecules (Notch1, Hes5, NeuroD1, β‐III‐Tubulin, synaptotagmin‐I and neurofilament‐M). Seven DSs and four transitional stages were defined from ED2 to ED12, when the basic OT cortical organization is established, along a spatial developmental gradient axis extending between a zone of maximal and a zone of minimal development. The table of DSs reveals that DSs do not only progress as a function of time but also display a spatially organized propagation along the developmental gradient axis. The complex and dynamic character of the OT development is documented by the fact that several DSs are simultaneously present at any ED or any embryonic stage. The table of DSs allows interpreting how developmental cell behaviors are temporally and spatially organized and explains how different DSs appear as a function of both time and space. The table of DSs provides a reference system to characterize the OT corticogenesis and to reliably compare observations made in different specimens. J. Morphol. 2011.

Sonic hedgehog (Shh)/Gli modulates the spatial organization of neuroepithelial cell proliferation in the developing chick optic tectum

BackgroundSonic hedgehog (Shh)/Gli pathway plays an important regulatory role on the neuroepithelial cells (NEc) proliferation in the dorsal regions of the developing vertebrate Central Nervous System. The aim of this paper was to analyze the effect of the Shh/Gli signaling pathway activation on the proliferation dynamics and/or the spatial organization of the NEc proliferation activity during early stages of the developing chick optic tectum (OT). In ovo pharmacological gain and loss of hedgehog function approaches were complemented with in vivo electroporation experiments in order to create ectopic sources of either Shh or Gli activator (GliA) proteins in the OT. NEc proliferating activity was analyzed at ED 4/4.5 by recording the spatial co-ordinates of the entire population of mitotic NEc (mNEc) located along OT dorsal-ventral sections. Several space signals (numerical sequences) were derived from the mNEc spatial co-ordinate records and analyzed by different standardized non-linear methods of signal analysis.ResultsIn ovo pharmacologic treatment with cyclopamine resulted in dramatic failure in the OT expansion while the agonist purmorphamine produced the opposite result, a huge expansion of the OT vesicle. Besides, GliA and Shh misexpressions interfere with the formation of the intertectal fissure located along the dorsal midline. This morphogenetic alteration is accompanied by an increase in the mNEc density. There is a gradient in the response of NEcs to Shh and GliA: the increase in mNEc density is maximal near the dorsal regions and decrease towards the OT-tegmental boundary. Biomathematical analyses of the signals derived from the mNEc records show that both Shh and GliA electroporations change the proliferation dynamics and the spatial organization of the mNEc as revealed by the changes in the scaling index estimated by these methods.ConclusionsThe present results show that the Shh/Gli signaling pathway plays a critical role in the OT expansion and modelling. This effect is probably mediated by a differential mitogenic effect that increases the NEc proliferation and modulates the spatial organization of the NEc proliferation activity.