Freeze-etching study of synaptosomes from rat cerebral cortex

Synaptosomes from rat cerebral cortex were studied using the freeze-etching technique. The intra-membranous structure of the pre- and postsynaptic membranes was examined. Particles with an electron-dense spot on their apex are reported from all fracture faces. Most probably these are related to transmembrane channels whose significance in the synaptic transmission is discussed.     

Cell lineage in the developing neural tube.

Acquisition of cell type specific properties in the spinal cord is a process of sequential restriction in developmental potential. A multipotent stem cell of the nervous system, the neuroepithelial cell, generates central nervous system and peripheral nervous system derivatives via the generation of intermediate lineage restricted precursors that differ from each other and from neuroepithelial cells. Intermediate lineage restricted neuronal and glial precursors termed neuronal restricted precursors and glial restricted precursors, respectively, have been identified. Differentiation is influenced by extrinsic environmental signals that are stage and cell type specific. Analysis in multiple species illustrates similarities between chick, rat, mouse, and human cell differentiation. The utility of obtaining these precursor cell types for gene discovery, drug screening, and therapeutic applications is discussed.     

frizzled 9 is expressed in neural precursor cells in the developing neural tube

The wnt signaling pathway has important functions in nervous system development. To better understand this process we have cloned and analyzed the expression of the wnt receptor, frizzled 9, in the developing nervous system in mouse, chick and zebrafish. The earliest expression of mouse frizzled 9 mRNA expression begins at E8.5 with expression throughout the entire rostral-caudal neuraxis. This early expression pattern within the neural tube appears to be conserved between chick and zebrafish. Expression becomes restricted to a ventral domain in the mouse ventricular zone at E11.5, a region specified to give rise to neurons and glia. Using a polyclonal antibody to MFZ9 further shows expression limited to neural restricted precursors cells.     

Cryofractographic study of intercellular junctions in the populations of agar-cultivated Bordetella pertussis

The characteristic feature of replicas obtained from the freeze-fractures of B. pertussis unfixed cultures developing on casein charcoal agar for 1-7 days is the associative growth of highly polymorphic cells, ensured by the ramified system of intercellular connections (IC) formed by the derivatives of the outer layers of the cell wall. This proves that the associative location of bacterial cells, linked by numerous IC, in the preparation is not the artefact appearing in the process of their chemical fixation. In replicas obtained from the freeze-fractures of B. pertussis cultures, previously fixed with glutaraldehyde, osmic acid and uranyl acetate, oval cells with the cytoplasm having a relatively homogeneous structure and with the smoothed-out three-layer cell wall prevail. As a rule, IC are limited to the sites of direct contacts between individual cells.     

Fine structure of the ependyma and intercellular junctions in the area postrema of the rat

Summary Ependymal cells and their junctional complexes in the area postrema of the rat were studied in detail by tracer experiments using horseradish peroxidase (HRP) and colloidal lanthanum and by freeze-etch techniques, in addition to routine electron microscopy. The ependyma of the area postrema is characterized as flattened cells possessing very few cilia, a moderate amount of microvilli, a well-developed Golgi apparatus and rough endoplasmic reticulum. Numerous vesicles or tubular formations with internal dense content were found to accumulate in the basal processes of ependymal cells; the basal process makes contact with the perivascular basal lamina. It is suggested that the dense material in the tubulovesicular formations is synthesized within the ependymal cell and discharged into the perivascular space. The apical junctions between adjacent ependymal cells display very close apposition, with a gap of 2–3 nm, but no fusion of adjacent plasma membranes; they thus represent a transitional form between the zonulae adhaerentes present in the ordinary mural ependyma and the zonulae occludentes in the choroidal epithelium. A direct intercommunication between the ventricular cerebrospinal fluid (CSF) and the blood vascular system indicates that a region exists lacking a blood-ventricular CSF barrier.     

Tubulobulbar complexes are intercellular podosome-like structures that internalize intact intercellular junctions during epithelial remodeling events in the rat testis

Tubulobulbar complexes are actin-related double-membrane projections that resemble podosomes in other systems and form at intercellular junctions in the seminiferous epithelium of the mammalian testis. They are proposed to internalize intact junctions during sperm release and during the translocation of spermatocytes through basal junction complexes between neighboring Sertoli cells. In this study we probe apical tubulobulbar complexes in fixed epithelial fragments and fixed frozen sections of rat and mouse testes for junction molecules reported to be present at apical sites of attachment (ectoplasmic specializations) between Sertoli cells and spermatids. The adhesion molecules nectin 2 (PVRL2), nectin 3 (PVRL3) and alpha 6 integrin (ITGA6) are present in the elongate parts of tubulobulbar complexes and concentrated at their distal ends. Tubulobulbar complexes contain cortactin (CTTN), a key component of podosomes, and vesicles at the distal ends of tubulobulbar complexes that contain junction molecules are related to early endosome antigen (EEA1). N-cadherin (CDH2), a protein reported to be present at ectoplasmic specializations, is not localized to these unique junctions or to tubulobulbar complexes but, rather, is primarily concentrated at desmosomes in basal regions of the epithelium. Our results are consistent with the conclusion that tubulobulbar complexes are podosome-like structures that are responsible for internalizing intact intercellular junctions during spermatogenesis.     

Various types of non-synaptic intercellular contacts in the developing brain of the rat

Peculiarities of ultrastructural organization and localization of early forms of avascular nonsynaptic types of junctions formed in 14-18-day-old rat embryos have been studied; cerebral structures different in their phylogenic relations (the sensomotor cortex and nucleus caudatus) are taken as an example. Five main types of nonsynaptic intercellular junctions have been revealed: desmosome-like, gap, symmetric, asymmetric and mixed junctions. They differ by their ultrastructural organization. These types of junctions make the main types of contacts: soma-somatic, dendro-somatic, dendro-dendritic, axo-somatic, axo-dendritic. Desmosomes form the greatest number of the contacts. The earliest and the most primitive are gap junctions; they, evidently, reflect functional activity of desmosome-like junctions. The mixed junctions, perhaps, reflect the developmental stages of the intercellular contacts of transition from one type of junctions into another. Localization peculiarities of the nonsynaptic intercellular contacts are demonstrated: glomerule-like formations, establishment of numerous contacts looking like a successive chain, and so on. For some other indices a longer period of intercellular contact formation in the nucleus caudatus is noted, comparing the sensomotor cortex, though the latter is a newer structural cerebral formation from the phylogenic point of view.     

Carboxylic esterases in developing myoneural junctions of rat striated muscle

Summary The distribution of acetylcholinesterase (AChE; E.C. 3.1.1.7), other cholinesterases (ns.ChE; E.C. 3.1.1.8) and eserine-resistant carboxylic esterases (ns. E; E.C. 3.1.1.1) was studied in the developing myoneural junction of the rat tibialis anterior muscle from the 16th intrauterine day onwards. Acetyl-and butyrylthiocholine were used as substrates for AChE and ns.ChE, and -naphthyl acetate for ns. E.Acetylcholinesterase was first visible in 18-day rat embryos, ns.ChE in 21-day embryos and ns. E in 1-day-old postnatal rats and thereafter. Both AChE and ns.ChE activities were localized at the level of the plasma membrane in the middle of the muscle fibres. In the early stages this area of activity had the appearance of a plate-like structure, which deepened to form a depression in the surface of the muscle fibre by the 2nd to the 4th postnatal day. About 5 days later subneural lamellaes appeared in this structure, and ramification and segmentation took place, their extent increasing concomitantly with the increase of enzyme activity. The adult pattern was attained by the age of one month. Precise localization of ns. E was not possible in the immature stages, mainly owing to the granularity of the reaction end-product. After the age of about 10 days, however, the distribution of the reaction end-product suggested a mainly presynaptic location. Other cholinesterases and ns. E, but not AChE, were detected in the neurilemma cells along nerve fibres. This neurilemmal enzyme activity gradually diminished after birth and was lost at about the age of 3 weeks.These observations demonstrate that the formation of the junction between the nerve and the muscle fibre takes place just before the first appearance of AChE activity in a sharply delineated area of the plasma membrane. The structural changes made apparent by the distribution of the reaction end-products are assumed to be linked to the spatial rearrangement of the synaptic membranes, seen in earlier electron microscopic studies.     

Norepinephrine inhibits intercellular coupling in rat cardiomyocytes by ubiquitination of connexin43 gap junctions

Gαq-stimulation reduces intercellular coupling within 10 min via a decrease in the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), but the mechanism is unknown. Here we show that uncoupling in rat cardiomyocytes after stimulation of α-adrenergic Gαq-coupled receptors with norepinephrine is prevented by proteasomal and lysosomal inhibitors, suggesting that internalization and possibly degradation of connexin43 (Cx43) is involved. Uncoupling was accompanied by increased Triton X-100 solubility of Cx43, which is considered a measure of the non-junctional pool of Cx43. However, inhibition of the proteasome and lysosome further increased solubility while preserving coupling, suggesting that communicating gap junctions can be part of the soluble fraction. Ubiquitination of Cx43 was also increased, and Cx43 co-immunoprecipitated with the ubiquitin ligase Nedd4. Conclusions: Norepinephrine increases ubiquitination of Cx43 in cardiomyocytes, possibly via Nedd4. We suggest that Cx43 is subsequently internalized, which is preceded by acquired solubility in Triton X-100, which does not lead to uncoupling per se.     

Norepinephrine inhibits intercellular coupling in rat cardiomyocytes by ubiquitination of connexin43 gap junctions

Abstract

Gα_q-stimulation reduces intercellular coupling within 10 min via a decrease in the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), but the mechanism is unknown. Here we show that uncoupling in rat cardiomyocytes after stimulation of α-adrenergic Gα_q-coupled receptors with norepinephrine is prevented by proteasomal and lysosomal inhibitors, suggesting that internalization and possibly degradation of connexin43 (Cx43) is involved. Uncoupling was accompanied by increased Triton X-100 solubility of Cx43, which is considered a measure of the non-junctional pool of Cx43. However, inhibition of the proteasome and lysosome further increased solubility while preserving coupling, suggesting that communicating gap junctions can be part of the soluble fraction. Ubiquitination of Cx43 was also increased, and Cx43 co-immunoprecipitated with the ubiquitin ligase Nedd4. Conclusions: Norepinephrine increases ubiquitination of Cx43 in cardiomyocytes, possibly via Nedd4. We suggest that Cx43 is subsequently internalized, which is preceded by acquired solubility in Triton X-100, which does not lead to uncoupling per se.     

BMP controls nitric oxide-mediated regulation of cell numbers in the developing neural tube

Balanced cell proliferation and cell death determines neural precursor cell numbers in early stages of neural tube (NT) development. We have previously shown that nitric oxide (NO) regulates cell numbers locally in the NT of eight to 12 somite embryos. Here, we demonstrate that bone morphogenetic protein-4 (BMP-4), which is expressed in the ectoderm and dorsal NT at these developmental stages, induces programmed cell death (PCD) and promotes entry into the S-phase, via nitric oxide synthase (NOS) activity. These effects can be reversed by BMP-4 antagonists, such as follistatin and noggin, or by specific NOS inhibitors, resulting in low NO levels that facilitate mitosis and reduce PCD. Ectopic BMP-4 induction of PCD is restricted to the dorsal NT, whereas promotion of the S-phase is evenly observed across the dorsal-ventral (D-V) axis. Prolonged exposure to either BMP-4 or NOS inhibitors, which results in high or low NO levels, respectively, causes NT defects. The results presented here throw new light on the BMP signaling pathway. The local presence of BMP-4 helps to regulate cell numbers in the developing NT by a NO-mediated pathway, which is essential for normal NT formation.