Formation of contacts between mast cells and sympathetic neurons in vitro

Summary Functional interactions between mast cells and peripheral nerves may occur at sites of association seen in vivo. To study the interactions, we developed a tissue culture model of murine sympathetic neurons co-cultured with rat basophilic leukaemia (RBL-2H3) cells (homologues of mucosal mast cells) or rat peritoneal mast cells. In co-cultures of up to 3 days, light microscopy identified neurite contacts with peritoneal mast cells or RBL-2H3 cells, but not with glial cells or fibroblasts. Electron microscopy confirmed membrane-membrane contact between neurites and RBL-2H3 cells. Time-lapse analysis of interactions between neurons and RBL-2H3 cells showed that 60–100% of the cells in a given field acquired neurite contact within 17 h. In matching control studies, there was no increase in the frequency of neurite contact with cells of the rat plasmacytoma line (YB2/0): these were not selected as targets, and contacts were broken if formed. Time-lapse records of the derivation of neurites from their path suggested a neurotropic effect of mast cells, with neurite contact ensuing when the intervening distance was less than 36±4 m. Once formed, contacts were invariably maintained throughout the period of examination (up to 72 h), in contrast to YB2/0 or fibroblast contacts. We conclude that neurons selectively form and maintain connections with cells representative of rat connective tissue-type and mucosal mast cells in vitro. Similar interactions in vivo could promote nerve/mast cell contacts, which may allow bidirectional communication between the nervous and immune systems.     

A real-time analysis of growth cone–target cell interactions during the formation of stable contacts between hippocampal neurons in culture

Mechanisms of cell-cell recognition and structural changes of growth cones (g.c.) and target membranes during contact formation are poorly understood. To examine these issues, we obtained a high magnification, realtime record of stale contact formation in cultured cells from the hippocampal CA1 area in the newborn rat. We used differential interference contrast (DIC) optics coupled to a video microscope for periods of over 24 h of continuous time-lapse recording. Our goal was to observe the sequential changes exhibited by afferent and target cells as they form a stable contact. Understanding the process of how stable contacts are made is important because such contacts are the first step in synapse formation. Four principal observations emerged from our study: (1) The target cell was receptive to a contact on a specific patch on its surface defined by the presence of lamellae and filopodia. This specific patch (named target site) was invariably present on the target cell surface before the time the growth cone arrived. (2) Stable adhesion between filopodia on the two cells initiated events leading to cell–cell contact formation. Specifically, the remaining filopodia on the growth cone and target cell were redirected toward the adhering filopodia, and the growth cone size decreased dramatically. (3) The axonal process then grew at a significantly accelerated rate (up to 50 times its baseline growth rate). (4) In addition, a number of observations were obtained on axonal turns towards the target cell, induction of target sites, and architectural remodelling of cells after the formation of a new contact. Our findings indicate that in this neuronal system, filopodia are the means used by cells to interact at stages prior to and during contact formation. We speculate that the molecules involved in cell recognition and the machinery that initiates contact formation are embedded in the fine structure of filopodia. Finally, our results provide possible clues as to some of the stages that may be involved in synapse formation in the mammalian central nervous system. © 1992 John Wiley & Sons, Inc.     

Formation of intercellular contacts in myogenesis

In myogenesis in vivo and in the muscle tissue culture certain intercellular junctions have been revealed; they differ in their ultrastructure and functions. For the stage of interaction between a myoblast with another myoblast contacts of adhesive type are distinctive: desmosomes and fasciae adherentes. They are necessary for adhesion of the cells with each other. Besides, gap and punctate contacts occur, serving for exchange of metabolites and electrical conjugation. At more advanced stages of fusion, when the myoblast gets into contact with the early muscle tubule, a bridge contact is observed, resembling the septal one, which is able to transform into a pentalayered (tight) junction. The latter type evidently participates in fusion of the membranes of the interacting cells.     

Formation of intermembrane contacts during myelinization

N.n. tibialis were investigated in 5--10-day-old rats, using electron microscopy. All the main types of membrane junctions were found in the area of forming nodose: hemi-desmosomal, serial desmosomal, continuous, septate, gap and tight junctions. The junctions were both of glio-glial and axon-glial type. These types may be stages of a unified process of membrane interaction. They are located in multiple loci and form intermediate dense myelin line upon merging. Such junctions result form aggregation and retraction of outer para-membrane electron dense material. The same mechanism of inner surface membrane coupling was observed in lamellipodia of lemmocytes. Thus "inside out" local junctions were formed. Merging of these junctions forms the main dense line of myelin. Consequently, compact myelin, tHus formed, should be considered as a gigantic complex membrane junction.     

A real-time analysis of growth cone-target cell interactions during the formation of stable contacts between hippocampal neurons in culture.

Mechanisms of cell-cell recognition and structural changes of growth cones (g.c.) and target membranes during contact formation are poorly understood. To examine these issues, we obtained a high magnification, real-time record of stable contact formation in cultured cells from the hippocampal CA1 area in the newborn rat. We used differential interference contrast (DIC) optics coupled to a video microscope for periods of over 24 h of continuous time-lapse recording. Our goal was to observe the sequential changes exhibited by afferent and target cells as they form a stable contact. Understanding the process of how stable contacts are made is important because such contacts are the first step in synapse formation. Four principal observations emerged from our study: (1) The target cell was receptive to a contact on a specific patch on its surface defined by the presence of lamellae and filopodia. This specific patch (named target site) was invariably present on the target cell surface before the time the growth cone arrived. (2) Stable adhesion between filopodia on the two cells initiated events leading to cell-cell contact formation. Specifically, the remaining filopodia on the growth cone and target cell were redirected toward the adhering filopodia, and the growth cone size decreased dramatically. (3) The axonal process then grew at a significantly accelerated rate (up to 50 times its baseline growth rate). (4) In addition, a number of observations were obtained on axonal turns towards the target cell, induction of target sites, and architectural remodelling of cells after the formation of a new contact. Our findings indicate that in this neuronal system, filopodia are the means used by cells to interact at stages prior to and during contact formation. We speculate that the molecules involved in cell recognition and the machinery that initiates contact formation are embedded in the fine structure of filopodia. Finally, our results provide possible clues as to some of the stages that may be involved in synapse formation in the mammalian central nervous system.     

Regions of putative acetylcholine receptors at synaptic contacts between neurons maintained in culture and subsequently fixed in solutions containing tannic acid

Summary Spinal cord neurons from 9-day chick embryos were maintained in culture for up to 35 days and then fixed in 4% cacodylate-buffered glutaraldehyde containing 2% tannic acid. After about 15 days in culture a small percentage of the synaptic specializations present were characterized by striking electron-dense striations averaging 15 nm in width, oriented perpendicular to the postsynaptic membrane. These structures increased in frequency with time in culture (to a maximum of about 10% of all synapses in the oldest cultures); they were asymmetrical, protruding approximately 8 nm into the synaptic cleft, and more deeply (approximately 15–18 nm), into the postsynaptic cytoplasm. On the basis of earlier work by Sealock (1980) they are interpreted as concentrations of acetylcholine receptors.Similar membrane differentiations were also seen associated with active-zone areas of a few presynaptic membranes, and the possibility that these represent presynaptic acetylcholine receptors is discussed. Additional observations reported are (1) the presence of striations resembling those seen at the postsynaptic membrane in the membranes of some postsynaptic vesicles, and (2) filamentous links between the striations and cytoskeletal elements of the postsynaptic cell.     

Highly permeable contacts between epithelial cells in culture]

Cultured epithelial cells producing monolayered sheets were used to study intercellular contacts permeable to sodium fluorescein. Cells in dense cultures were more capable of forming permeable junctions than cells of sparse cultures. In addition, the standard microelectrode technique revealed some differences in cellular membrane potentials in dense and sparse cultures. A possible correlation is discussed between intercellular contact formation and other features of cells depending on cell culture density.     

The forms of the intercellular contacts of Leishmania in culture]

Two kinds of intercellular interactions have been observed in cultures of 12 investigated Leishmania species (L. major, L. tropica, L. donovani, L. infantum, L. sp. ZMA, L. mexicana, L. hertigi, L. braziliensis, L. tarentolae, L. adleri, L. gymnodactyli, L. gulickae). The first kind looks as adhesion of two specimens with their fore-ends. This way is characteristic of promastigotes of different morphotypes as well as of the interphase and dividing organisms to be most frequently seen in L. mexicana and L. gymnodactyli, and in both dark and lucid forms. The second kind of intercellular interactions involves a coupled adhesion of morphologically similar promastigotes with free ends of flagella. It is most characteristic of L. gymnodactyli and specially of dark promastigotes. Proves are provided that both the kinds of cell interactions are not associated with cell division, that they may only partially be connected with the phenomenon of rosette formation, and that they represent different phenomena. It is supposed that the intercellular contacts with the fore-ends may reflect gene exchanges in two partners, with a possible involvement of the kinetoplast DNA.     

Vagus nerve stimulation preferentially induces Fos expression in nitrergic neurons of rat esophagus

To identify neurochemical phenotypes of esophageal myenteric neurons synaptically activated by vagal preganglionic efferents, we immunohistochemically detected the expression of Fos, an immediate early gene product, in whole-mount preparations of the entire esophagus of rats following electrical stimulation of the vagus nerves. When electrical stimulation was applied to either the cervical left (LVN) or right vagus nerve (RVN), neurons with nuclei showing Fos immunoreactivity (IR) were found to comprise approximately 10% of the total myenteric neurons in the entire esophagus. These neurons increased from the oral toward the gastric end of the esophagus, with the highest frequency in the abdominal portion of the esophagus. A significant difference was not found in the number of Fos neurons between the LVN-stimulated and RVN-stimulated esophagus. Double-immunolabeling showed that nitric oxide synthase (NOS)-IR occurred in most (86% and 84% in the LVN-stimulated and RVN-stimulated esophagus, respectively) of the Fos neurons in the entire esophagus. Furthermore, the stimulation of either of the vagus nerves resulted in high proportions (71%-90%) of Fos neurons with NOS-IR, with respect to the total Fos neurons in each segment, in the entire esophagus. However, a small proportion (8% and 7% in the LVN-stimulated and RVN-stimulated esophagus, respectively) of the Fos neurons in the esophagus exhibited choline acetyltransferase (ChAT)-IR. The occurrence-frequency of Fos neurons with ChAT-IR was less than 4% of the total Fos neurons in any segment of the LVN-stimulated and RVN-stimulated esophagus. Some of the Fos neurons with ChAT-IR appeared to be innervated by numerous varicose ChAT-positive nerve terminals. The present results showing that electrical stimulation of the vagus nerves induces a high proportion of Fos neurons with NOS-IR suggests the preferential activation of NOS neurons in the esophagus by vagal preganglionic efferents. This connectivity between the vagal efferents and intrinsic nitrergic neurons might be involved in inhibitory actions on esophageal motility.This study was supported by Grant-in Aids for Scientific Research from Ministry of Education, Sports, and Culture of Japan to H.K. (no. 15500236) and to M.K. (no. 14570065).     

Desmosome-like contacts of Mauthner neurons as targets of scorpion venom

Desmosome-like contacts (DLC) in afferent chemical synapses of the Mauthner cells (MC) were investigated after application of low and high molecular mass peptide fractions 6 and 9, correspondingly, from the Central Asiatic black scorpion Orthochirus scrobiculosus. Besides, the DLC were examined in condition of a training induced morpho-functional stability of the MC (adaptation) mediated by transformation of actin monomers into polymers. In addition, the structure of DLC was studied after cytochalasin application which disrupts F-actin. Fraction 6 was shown to increase the length of DLC and osmiophily of fibrous material. Similar changes in DLC were caused by adaptation. Fraction 9 decreased the osmiophily of the fibrous material, made DLC asymmetric, but did not influence their length. Similar changes in DLC were seen also after cytochalasin D application. Taking into account our previous data on the role of F-actin in the MC functioning, which were obtained following specific pharmacological treatments, the similarity of ultrastructural changes in DLC after both adaptation and fraction 6 application, on the one hand, and after both cytochalasin D and fraction 9 application, on the other one, enabled us to suggest that these fractions may contain peptides able to exert influence of the actin cytoskeleton.     

Scanning electron microscopy of nerve-muscle contacts in embryonic cell culture.

Scanning electron microscopy (SEM) of cell cultures of dissociated nerve and muscle from chick embryos has shown that developing muscle fibers can be contacted at many sites by one or more than one neuron, and that a single nerve can send branches to several myofibers. At these contact regions of nerve with muscle, the neurons send out terminal or lateral sprouts with fine tips which initially lack terminal swellings, but later acquire small “bouton”-like structures in contact with the sarcolemma, which resemble embryonic synapses. At these points, the sarcolemma does not appear to differ in ultrastructure from other surface regions of the myofiber. Transmission electron microscopy (TEM) has revealed the presence of both electron lucent and dense-cored vesicles at some nerve terminals. However, fluorescence histochemistry (Falck-Hillarp technique) failed to detect the presence of catecholamines in these cultures. The SEM pictures at substantially higher resolutions than the light microscope, and the enhanced three dimensional perspective of this technique, provide additional information about the developmental morphology of the nerve-muscle cell culture system. The results are correlated with previous findings by light microscopy, TEM and electrophysiology, and discussed in relationship to proposed innervation processes of skeletal muscle fibers in vivo.     

Formation of multiple-oocyte follicles in culture

Basement membranes are found in every organ of the body. They provide structure and a selective filter for molecules. The ovary is no different with the follicular basal lamina (FBL) separating the granulosa and theca cells, facilitating regulation of the changing follicular environment providing appropriate conditions for the developing oocyte. The FBL is modified in C1galt1 Mutant mice (C1galt1 ^FF:ZP3Cre) resulting from oocyte-specific deletion of C1galt1. Changes in the FBL lead to follicles joining to generate multiple-oocyte follicles (MOFs); where two or more oocytes are contained within a single follicle. This study aimed to determine if single-oocyte follicles could join in culture to become MOFs by co-culturing preantral follicles from Control or Mutant mice. Co-cultured follicles from both Control and Mutant follicles could superficially fuse (73% of Control follicle pairs; 84% of Mutant). Confocal microscopy revealed alterations in the organization of the space between follicles but was unable to discern MOFs. When co-cultured follicle pairs were embedded, sectioned and stained with haematoxylin, it was revealed that MOFs had formed from 50% of Mutant follicle pairs but none from Control follicle pairs. In conclusion, MOFs can form from C1galt1 Mutant follicles in culture and this model is a useful tool to elucidate the role of the oocyte in follicle development and the generation and function of the FBL. Furthermore, understanding the relationship between oocyte function and FBL generation will likely provide insight into optimizing conditions for follicle culture, which is important for fertility treatments and ART.     

Modulation of neural commitment by changes in target cell contacts in Pleurodeles waltl

In amphibian development, neural structures arise from the presumptive ectoderm at the gastrula stage by an inductive interaction with the chordamesoderm. It has been previously reported that early gastrula presumptive ectoderm can be neuralized when it is dissociated into single cells. A similar result is reported here with regard to Pleurodeles waltl presumptive ectoderm. Using this experimental model system we demonstrate: first, that neuronal and glial lineages can be specified from the presumptive ectoderm without any intervention of the natural inducing tissue; and second, that whereas rupture of cell-cell contacts evoked neural induction, dissociation immediately followed by reaggregation reduces the neuralizing response, pointing toward an active role played by cell-cell contacts of presumptive ectodermal cells in the modulation of neural commitment.     

Laser microirradiation of cerebellar neurons in culture

Electrophysiological and ultrastructural effects of focused laser radiation on neurons from neonatal rat cerebellum in tissue culture are reported. Action potentials were elicited by an extracellular current pulse train. The stimulator voltage required for half-maximum response frequency was measured as a function of the energy delivered by a single laser pulse. Above a “threshold” laser energy, the cell response to stimulation became negligible for all stimulator voltages. Electron micrographs of cells revealed that the mitochondria are preferentially damaged at an energy comparable to the electrophysiological threshold. The damaged mitochondria showed swollen matrix space and disrupted cristae membranes. Higher laser energies resulted in damage to other cytoplasmic structures. The results are consistent with a model that assumes that light interaction with the nerve cells proceeds by local heating of the mitochondria and nearby structures and leads to an increased conductance of the membrane to some ionic species.     

Myelination of rodent hippocampal neurons in culture

Axons of various hippocampal neurons are myelinated mainly postnatally, which is important for the proper function of neural circuits. Demyelination in the hippocampus has been observed in patients with multiple sclerosis, Alzheimer's disease or temporal lobe epilepsy. However, very little is known about the mechanisms and exact functions of the interaction between the myelin-making oligodendrocytes and the axons within the hippocampus. This is mainly attributable to the lack of a system suitable for molecular studies. We recently established a new myelin coculture from embryonic day (E) 18 rat embryos consisting of hippocampal neurons and oligodendrocytes, with which we identified a novel intra-axonal signaling pathway regulating the juxtaparanodal clustering of Kv1.2 channels. Here we describe the detailed protocol for this new coculture. It takes about 5 weeks to set up and use the system. This coculture is particularly useful for studying myelin-mediated regulation of ion channel trafficking and for understanding how neuronal excitability and synaptic transmission are regulated by myelination.     

Hypoxic vasodilation in porcine coronary artery is preferentially inhibited by organ culture

Hypoxia (95% N₂-5%CO₂) elicits an endothelium-independent relaxation(45-80%) in freshly dissected porcine coronary arteries. Pairedartery rings cultured at 37°C in sterile DMEM (pH ~7.4) for 24 h contracted normally to KCl or 1 µM U-46619. However, relaxation inresponse to hypoxia was sharply attenuated compared with control (fresharteries or those stored at 4°C for 24 h). Hypoxicvasorelaxation in organ cultured vessels was reduced at both high andlow stimulation, indicating that both Ca²⁺-independent andCa²⁺-dependent components are altered. In contrast,relaxation to G-kinase (sodium nitroprusside) or A-kinase (forskolinand isoproterenol) activation was not significantly affected by organculture. Additionally, there was no difference in relaxation afterwashout of the stimulus, indicating that the inhibition is specific toacute hypoxia-induced relaxation. Simultaneous force and intracellularcalcium concentration ([Ca²⁺]_i) measurementsindicate the reduction in [Ca²⁺]_i concomitantwith hypoxia at low stimulus levels in these tissue is abolished byculture. Our results indicate that organ culture at 37°C specificallyattenuates hypoxic relaxation in vascular smooth muscle by alteringdynamics of [Ca²⁺]_i handling and decreasing aCa²⁺-independent component of relaxation. Thus organculture can be a novel tool for investigating the mechanisms ofhypoxia-induced vasodilation.

     

The effect of iron on mammalian cortical neurons in culture

We added iron in the ferric form to predominantly neuronal, cortical cell cultures, and determined clonazepam-displaceable [³H]diazepam binding, choline acetyltransferase activity, high-affinity [³H]GABA uptake, and glutamic acid decarboxylase activity. Chronic exposure (14 days) to low concentrations (0.01, 0.04, and 0.1 g/ml) of added ferric iron resulted in a significant decrease in each of the measures studied.     

Crystals of TELSAM–target protein fusions that exhibit minimal crystal contacts and lack direct inter-TELSAM contacts

While conducting pilot studies into the usefulness of fusion to TELSAM polymers as a potential protein crystallization strategy, we observed novel properties in crystals of two TELSAM–target protein fusions, as follows. (i) A TELSAM–target protein fusion can crystallize more rapidly and with greater propensity than the same target protein alone. (ii) TELSAM–target protein fusions can be crystallized at low protein concentrations. This unprecedented observation suggests a route to crystallize proteins that can only be produced in microgram amounts. (iii) The TELSAM polymers themselves need not directly contact one another in the crystal lattice in order to form well-diffracting crystals. This novel observation is important because it suggests that TELSAM may be able to crystallize target proteins too large to allow direct inter-polymer contacts. (iv) Flexible TELSAM–target protein linkers can allow target proteins to find productive binding modes against the TELSAM polymer. (v) TELSAM polymers can adjust their helical rise to allow fused target proteins to make productive crystal contacts. (vi). Fusion to TELSAM polymers can stabilize weak inter-target protein crystal contacts. We report features of these TELSAM–target protein crystal structures and outline future work needed to validate TELSAM as a crystallization chaperone and determine best practices for its use.