Gap junction-mediated bidirectional signaling between human fetal hippocampal neurons and astrocytes.

Gap junctions are clusters of intercellular channels that connect the interiors of coupled cells. In the brain, gap junctions function as electrotonic synapses between neurons and as pathways for the exchange of metabolites and second-messenger molecules between glial cells. Astrocytes, the most abundant glial cell type coupled by gap junctions, are intimately involved in the active control of neuronal activity including synaptic transmission and plasticity. Previous studies have suggested that astrocytic-neuronal signaling may involve gap junction-mediated intercellular connections; this issue remains unresolved. In this study, we demonstrate that second-trimester human fetal hippocampal neurons and astrocytes in culture are coupled by gap junctions bidirectionally; we show that human fetal neurons and astrocytes express both the same and different connexin subtypes. The formation of functional homotypic and heterotypic gap junction channels between neurons and astrocytes may add versatility to the signaling between these cell types during human hippocampal ontogeny; disruption of such signaling may contribute to CNS dysfunction during pregnancy.

Gap junctions: the "kiss of death" and the "kiss of life".

Cells expressing herpes simplex-thymidine kinase (HSV-tk) can be killed "in vitro" within 5 days of treatment with 20 microM ganciclovir (GCV) and transmit this toxicity to adjacent cells lacking HSV-tk; this phenomenon was termed "bystander effect" or "kiss of death". On testing a large number of cell lines in vitro, a wide range of sensitivity to GCV-mediated bystander killing has been reported. Although intercellular transfer of GCV metabolites through gap junction channels seems to be a likely mechanism for the "kiss of death", some studies suggest that other pathways may contribute to induced apoptosis of neighboring cells. To further investigate the mechanism underlying cell death mediated by HSV-tk and to evaluate the efficacy of gap junction channels formed by different connexins in this process, we have stably transfected a virtually uncoupled mouse neuroblastoma cell line (N2A cells) with different connexin-types expressed by neural cells (Cx32, Cx37, Cx40, Cx43) and co-cultured these cells with N2A cells stably transfected with Cx37 and HSV-tk. Here, we confirm our previous studies and those of others that the extent of cell death and sensitivity to GCV depend on the degree of connexin expression in transfectants. Further, we show that the bystander effect also depends on which connexin is expressed; reported disparities regarding the extent of GCV-mediated cellular apoptosis are likely due both to the degree of functional coupling and the type of connexin expressed. These results support the notion that gap junction hemichannels formed of certain connexins are more likely than others to pair functionally with Cx37, and suggest co-transfection strategies that might prove effective in sensitizing tumor cell populations to GCV. In addition, potential applications are discussed for use of the "good Samaritan effect", a mechanism by which bystander cells have been suggested to prevent cytotoxicity.

Identification of mayaro virus nucleocapsid protein in nucleus of Aedes albopictus cells.

The modifications in the pattern of nuclear proteins of Aedes albopictus cells in response to Mayaro virus infection were analysed early and late after infection. The viral capsid (C) protein of 34 kDa (p34) could be detected in the nuclear compartment 4 h after infection, soon after its synthesis in the cytoplasm. In addition to p34, a group of high molecular weight proteins was also present in this compartment late after infection. The exposition of infected cells to supra optimal temperature of growth modifies significantly the pattern of nuclear proteins. However, the stress condition does not inhibit the transport of p34 to the nucleus. The transport of proteins into nuclei was also followed under "in vitro' conditions by incubating radiolabeled post-mitochondrial extract of infected cells with unlabeled nuclei. Under these conditions, as observed "in vivo', a specific transport of viral C protein and of a group of proteins of high molecular weight to the nuclei was also detected. These results indicate that Mayaro virus infection modifies the nuclear protein pattern in invertebrate cells.

Characterization of two populations of ectopic cells isolated from the hearts of NGF transgenic mice.

We have observed sympathetic hyperinnervation and hyperplasia of an unknown cell population within the base of the hearts from transgenic mice in which nerve growth factor (NGF) expression was targeted to cardiac tissue (Hassankhani et al., 1993, 1995). To characterize this ectopic cell population, cells from the base of hearts of 3- to 4-week-old transgenic mice were dissociated and established in tissue culture, and their properties were analyzed using morphological, immunocytochemical, and physiological techniques. Morphological studies of the ectopic cells revealed the temporal expression of two different cellular phenotypes: (i) a spindle-shaped cell type, present by 1 to 2 days in vitro (DIV) and (ii) cells with a fiber-like morphology, detected by 7 DIV. In contrast to transgenic cardiac myocytes that express immunoreactivity to antibodies against sarcomeric myosin, beat spontaneously (approximately 60/min), and are electrically excitable, the ectopic cell types did not stain with an antibody against sarcomeric myosin, never showed contractile activity, and did not manifest membrane excitability. Moreover, these two types of ectopic cells demonstrated other distinctive characteristics. The spindle-shaped subpopulation typically formed small clusters of cells that were immunostained by antibodies to GFAP, vimentin, and low-affinity NGF receptor (LNGFR) and demonstrated a low incidence of dye coupling (30%), whereas the fiber-like cells aligned themselves along their long axes, immunostained for gp140trkA, LNGFR, and anti alpha-smooth muscle actin and showed extensive (100%) and diffuse dye spread to numerous other contiguous fiber-like cells. Dye spread was not observed between the adjacent fiber-like and the spindle-shaped cells and was reversibly blocked between homologous cells after treatment with halothane, a gap junction channel blocking agent. In comparison to cardiac myocytes, macroscopic junctional conductance of the spindle-shaped cells was more voltage-sensitive. At the single channel level, unitary junctional conductances of approximately 60 pS were predominant. Overall, these results indicate that these ectopic cells are likely derived from neural crest. The spindle-shaped cells appear to be immature Schwann cells, whereas the fiber-like cells may be related to the ectomesenchymal cells contributing to the morphogenesis of the cardiac outflow tract.

Differentiation of hippocampal progenitor cells in vitro: temporal expression of intercellular coupling and voltage- and ligand-gated responses.

Mechanisms regulating the expression of intercellular coupling, development of membrane excitability, and cellular responsiveness to neurotransmitters during neuronal ontogeny are largely unknown. To define the temporal relationship among these properties during neurogenesis, murine embryonic hippocampal progenitor cells immortalized with a temperature-sensitive allele of the SV40 large T-antigen were examined during successive stages of neuronal differentiation in vitro using patch clamp, dye coupling, and Ca2+ imaging techniques. Electrotonic and dye coupling between untreated neuroblasts were frequent in cells maintained at the temperature (39 degrees C) nonpermissive for T-antigen expression. However, as neuroblasts differentiated into neurons under the influence of interleukin-7 added alone or concurrently with transforming growth factor-alpha after basic fibroblast growth factor, both junctional conductance and the extent of dye coupling progressively decreased. Voltage-dependent inward currents were present within 2 to 6 days after differentiating treatments began. During intermediate developmental stages (3 to 5 days in culture), cells became responsive to GABA (> or = 100 microM) but not to glutamate, glycine, or to acetylcholine (< or = 1 mM), as indicated by [Ca2+]i measurements and patch clamp recordings. In contrast, voltage- and ligand-gated responses but not electronic coupling were frequently observed in mature neuronal primary cultures. Together, these results indicate that certain cytokines may orchestrate the progressive expression of functional neuronal phenotypes in vitro, in which the gradual disappearance of intercellular coupling parallels the onset of voltage-dependent responses and both of which precede the expression of neurotransmitter chemosensitivity.