Loss of Ephaptic Contacts in the Murine Thalamus during Osmotic Demyelination Syndrome.

BACKGROUND AND AIM: A murine model mimicking osmotic demyelination syndrome (ODS) revealed with histology in the relay posterolateral (VPL) and ventral posteromedial (VPM) thalamic nuclei adjoined nerve cell bodies in chronic hyponatremia, amongst the damaged 12 h and 48 h after reinstatement of osmolality. This report aims to verify and complement with ultrastructure other neurophysiology, immunohistochemistry, and molecular biochemistry data to assess the connexin-36 protein, as part of those hinted close contacts.This ODS investigation included four groups of mice: Sham (NN; n = 13), hyponatremic (HN; n = 11), those sacrificed 12 h after a fast restoration of normal natremia (ODS12h; n = 6) and mice sacrificed 48 h afterward, or ODS48 h (n = 9). Out of these, thalamic zones samples included NN (n = 2), HN (n = 2), ODS12h (n = 3) and ODS48h (n = 3). RESULTS: Ultrastructure illustrated junctions between nerve cell bodies that were immunolabeled with connexin36 (Cx36) with light microscopy and Western blots. These cell's junctions were reminiscent of low resistance junctions characterized in other regions of the CNS with electrophysiology. Contiguous neurons showed neurolemma contacts in intact and damaged tissues according to their location in the ODS zones, at 12 h and 48 h post correction along with other demyelinating alterations. Neurons and ephaptic contact measurements indicated the highest alterations, including nerve cell necrosis in the ODS epicenter and damages decreased toward the outskirts of the demyelinated zone. CONCLUSION: Ephapses contained C × 36between intact or ODS injured neurons in the thalamus appeared to be resilient beyond the core degraded tissue injuries. These could maintain intercellular ionic and metabolite exchanges between these lesser injured regions and, thus, would partake to some brain plasticity repairs.

PUFAs supplementation affects the renal expression of pannexin 1 and connexins in diabetic kidney of rats.

In diabetic nephropathy (DN), intercellular communication is disrupted. Connexins (Cx) have a crucial role in that process. Dietary ratios and supplementation with polyunsaturated fatty acids (PUFAs) can alleviate diabetic complications and cause alterations in Cx levels. Although pannexins (Panx) share similarities with members of the Cx family, their function in diabetic nephropathy has still not been fully determined. We studied the influence of PUFA supplementation on the immunoexpression of Px1 and Cx family members in diabetic kidneys of rats. Four groups of rats in experimental DM1 model were supplemented with different dietary n-6/n-3 ratios; ≈7 in control (C) and diabetic groups (STZ), ≈ 60 in the STZ + N6 group and ≈ 1 (containing 16% EPA and 19% DHA) in the STZ + N3 group. Immunoexpression of Cx40, Cx43, Cx45 and Panx1 was evaluated in the renal tissue of diabetic rats using immunohistochemistry. Diabetes significantly decreased the protein expression of Cx40 and Cx43 and increased Panx1 protein expression in the renal cortex (p < 0.05-p < 0.01). There was a significant impact of diet on Cx and Panx1 immunoexpression. Dietary supplementation with a high n-6/n-3 ratio downregulated the protein expression of Cx45 and Panx1 in diabetic rats (p < 0.05-p < 0.01), while Cx43 immunoexpression was increased in diabetic rats fed with high and low n-6/n-3 ratios (p < 0.01-p < 0.001). Hyperglycaemic conditions in DN interfere with cell-to-cell communication and disturb the connection between cells and their immediate environment due to variations in connexin and pannexin immunoexpression. These variations can be regulated by PUFA dietary intake, suggesting their beneficial effect and possible therapeutic option.

Unexpected effect of the vehicle (grain ethanol) of homeopathic FSH on the in vitro survival and development of isolated ovine preantral follicles.

The aims of this study were to investigate the effects of medium replacement system (experiment I) and of FSH presentations (homeopathic - FSH 6cH and allopathic FSH - rFSH; experiment II) on the in vitro development, hormone production and gene expression of isolated ovine preantral follicles cultured for 6 days. In experiment I, secondary follicles were cultured in the α-MEM+ supplemented with FSH 6cH (0.05 fg/ml) or recombinant bovine FSH (100 ng/ml) without/with daily medium addition. The homeopathic FSH treatments with/without medium addition improved (p < .05) follicular development compared to rFSH100 treatment without addition. FSH 6cH with addition showed the highest (p < .05) estradiol production. To verify whether the effects of homeopathic FSH were not due to its vehicle, experiment II was performed. The α-MEM+ was supplemented or not with alcohol (0.2% grain ethanol, v/v), FSH 6cH or rFSH100 with daily medium addition. Surprisingly, we found that all treatments improved follicular development compared to the α-MEM+ (p < .05). Moreover, homeopathic FSH was similar to the other treatments including its vehicle. In conclusion, its vehicle (ethanol) causes the effect of homeopathic FSH on in vitro development of isolated ovine preantral follicles.

Modulation of pulmonary alveolar type II cell phenotype and communication by extracellular matrix and KGF.

The alveolar epithelium consists of two cell types, alveolar type I (AT1) and alveolar type II (AT2) cells. We have recently shown that 7-day-old cultures of AT2 cells grown on a type I collagen/fibronectin matrix develop phenotypic characteristics of AT1 cells, display a distinct connexin profile, and coordinate mechanically induced intercellular Ca(2+) changes via gap junctions (25). In this study, we cultured AT2 cells for 7 days on matrix supplemented with laminin-5 and/or in the presence of keratinocyte growth factor. Under these conditions, cultured AT2 cells display AT2 type morphology, express the AT2-specific marker surfactant protein C, and do not express AT1-specific cell marker aquaporin 5, all consistent with maintenance of AT2 phenotype. These AT2-like cells also coordinate mechanically induced intercellular Ca(2+) signaling, but, unlike AT1-like cells, do so by using extracellular nucleotide triphosphate release. Additionally, cultured cells that retain AT2 cell-specific markers express connexin profiles different from cultured cells with AT1 characteristics. The parallel changes in intercellular Ca(2+) signaling with cell differentiation suggest that cell signaling mechanisms are an intrinsic component of lung alveolar cell phenotype. Because lung epithelial injury is accompanied by extracellular matrix and growth factor changes, followed by extensive cell division, differentiation, and migration of AT2 progenitor cells, we suggest that similar changes may be vital to the lung recovery and repair process in vivo.

Connexin 26 expression and extensive gap junctional coupling in cultures of GT1-7 cells secreting gonadotropin-releasing hormone.

Gap junctions (GJs) are transmembrane channels that permit rapid intercellular transit of various small molecules including ions, second messengers and metabolites. GJs promote communication and coordinated activity between coupled neurons, and may help facilitate the synchronous release and pulsatile secretion of neurohormones. A previous study using GnRH-secreting GT1-7 cells reported that connexin 26 was the major GJ subunit present, and that about 20% of the cultured cells engaged in GJ coupling as assayed by fluorescence recovery after photobleaching of 5,6-carboxyfluorescein diacetate (MW 460 D). To reassess GJ connectivity with a more permeant probe, we grew GT1-7 cells to 70% confluency on Matrigel-coated glass coverslips and microinjected Neurobiotin(TM) (MW 322 D) into single cells. Dye was allowed to diffuse for 30 min before cultures were fixed, and subsequently immunostained for Neurobiotin with 3,3'-diaminobenzidine HCl and examined by light microscopy. Dye coupling between 2 or more GT1-7 cells was observed after 75% of all microinjections. Connectivity involved the somata and neurites of an average of 6.6 +/- 2.0 adjoining cells, but in one instance was seen in a group of 32 GT1-7 neighbors. Western blotting and immunofluorescence staining confirmed that connexin 26 was the predominant GJ subunit expressed by GT1-7 cultures. Our results using Neurobiotin suggest these GJ channels may be smaller than anticipated. In addition, functional GJ connectivity between subconfluent GT1-7 cells is more extensive than previously reported, occurring with higher frequency and coupling significantly greater numbers of cultured cells. Since cAMP, IP3, and Ca(2+) are able to pass through GJs and can elicit secretion of GnRH by GT1 cell cultures, GJs may play an important role in the coordination and synchronization of GnRH release.

Changes in immunostaining of inner ears after antigen challenge into the scala tympani.

To study the mechanisms of immune responses and immune injuries in inner ears, labyrinthitis was induced by inoculation of keyhole limpet hemocyanin (KLH) into the scala tympani of systemically sensitized guinea pigs. Inner ears were then immunostained for KLH, immunoglobulin G (IgG), albumin, connexin26 (Cx26), and sodium-potassium adenosine triphosphate (Na,K-ATPase). Inflammatory cells containing KLH were observed in the scala tympani and in the collecting venule of the spiral modiolar vein (SMV). Spiral ligament, spiral limbus, and blood vessels including the SMV were diffusely positive for IgG and albumin. Immunoreactivity for Cx26 and Na,K-ATPase was decreased compared with the normal ears in the fibrocytes of the spiral ligament. These results suggest that inflammatory cells and blood constituents could extravasate into the cochlea from blood vessels and that fibrocyte damage in the spiral ligament could cause cochlear dysfunction.

Identification of gap junctional connexin-32 mRNA and protein in gonadotropin-releasing hormone neurons of the female rat.

Pulsatile gonadotropin-releasing hormone (GnRH) release from the median eminence is critical for the appropriate function of the pituitary gonadotropes and for the generation of a preovulatory gonadotropin surge. The mechanisms by which many GnRH axon terminals are synchronized to release GnRH in a coordinated fashion into the capillaries of the primary plexus are unknown as are the anatomical sites at which the regulation of GnRH neurons takes place. While many neurotransmitters have been shown to influence GnRH release, it is not clear if such neurotransmitters regulate GnRH neurons directly through synaptic interactions or through intermediate neurons. An alternative mechanism of interneuronal communication is provided by gap junctions which allow a rapid, bidirectional exchange of signals. In order to explore if GnRH neurons synthesize the appropriate proteins to form gap junctions with adjacent cells we used double immunohistochemistry for GnRH and connexins-26, -32 or -43 as well as dual in situ hybridization to identify GnRH mRNA and connexin-32 mRNA. The results show that all GnRH neurons contain connexin-32 immunoreactive puncta at their perikarya and, occasionally, at their axon terminals in the median eminence while connexin-26 and -43 immunoreactivity was absent in GnRH neurons. In addition, connexin-32 mRNA was detected in GnRH mRNA containing neurons. However, gap junctional connections between adjacent GnRH neurons were not observed. The data suggest that gap junctional coupling of GnRH neurons with neighboring non-GnRH containing cells may occur and may represent a mechanism by which GnRH neurons can be synchronized or by which hormonal or neurotransmitter signals can be conveyed to the GnRH neurons.