Activity-related calcium dynamics in motoneurons of the nucleus hypoglossus from mouse

A quantitative analysis of activity-related calcium dynamics was performed in motoneurons of the nucleus hypoglossus in the brain stem slice preparation from mouse by simultaneous patch-clamp and microfluorometric calcium measurements. Motoneurons were analyzed under in vitro conditions that kept them in a functionally intact state represented by rhythmic, inspiratory-related bursts of excitatory postsynaptic currents and associated action potential discharges. Bursts of electrical activity were paralleled by somatic calcium transients resulting from calcium influx through voltage-activated calcium channels, where each action potential accounted for a calcium-mediated charge influx around 2 pC into the somatic compartment. Under in vivo conditions, rhythmic-respiratory activity in young mice occurred at frequencies up to 5 Hz, demonstrating the necessity for rapid calcium elevation and recovery in respiratory-related neurons. The quantitative analysis of hypoglossal calcium homeostasis identified an average extrusion rate, but an exceptionally low endogenous calcium binding capacity as cellular parameters accounting for rapid calcium signaling. Our results suggest that dynamics of somatic calcium transients 1) define an upper limit for the maximum frequency of respiratory-related burst discharges and 2) represent a potentially dangerous determinant of intracellular calcium profiles during pathophysiological and/or excitotoxic conditions.     

Identification and characterization of a Neospora caninum microneme-associated protein (NcMIC4) that exhibits unique lactose-binding properties

Microneme proteins have been shown to play an important role in the early phase of host cell adhesion, by mediating the contact between the parasite and host cell surface receptors. In this study we have identified and characterized a lectin-like protein of Neospora caninum tachyzoites which was purified by alpha-lactose-agarose affinity chromatography. Upon separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this lactose-binding protein migrated at 70 and 55 kDa under reducing and nonreducing conditions, respectively. Immunofluorescence and immunogold electron microscopy with affinity-purified antibodies showed that the protein was associated with the tachyzoite micronemes. Mass spectrometry analyses and expressed sequence tag database mining revealed that this protein is a member of the Neospora microneme protein family; the protein was named NcMIC4 (N. caninum microneme protein 4). Upon two-dimensional gel electrophoresis, NcMIC4 separated into seven distinct isoforms. Incubation of extracellular parasites at 37 degrees C resulted in the secretion of NcMIC4 into the medium as a soluble protein, and the secreted protein exhibited a slightly reduced M(r) but retained its lactose-binding properties. Immunofluorescence was used to investigate the temporal and spatial distribution of NcMIC4 in tachyzoites entering their host cells and showed that reexpression of NcMIC4 took place 30 min after entry into the host cell. Incubation of secreted fractions and purified NcMIC4 with Vero cells demonstrated binding of NcMIC4 to Vero cells as well as binding to chondroitin sulfate A glycosaminoglycans.     

Cell domain-dependent changes in the glutamatergic and GABAergic drives during epileptogenesis in the rat CA1 region

An increased ratio of the glutamatergic drive to the overall glutamatergic/GABAergic drive characterizes the chronic stage of temporal lobe epilepsy (TLE), but it is unclear whether this modification is present during the latent period that often precedes the epileptic stage. Using the pilocarpine model of TLE in rats, we report that this ratio is decreased in hippocampal CA1 pyramidal cells during the early phase of the latent period (3–5 days post pilocarpine). It is, however, increased during the late phase of the latent period (7–10 days post pilocarpine), via cell domain-dependent alterations in synaptic current properties, concomitant with the occurrence of interictal-like activity in vivo . During the late latent period, the glutamatergic drive was increased in somata via an enhancement in EPSC decay time constant and in dendrites via an increase in EPSC frequency and amplitude. The GABAergic drive remained unchanged in the soma but was decreased in dendrites, since the drop off in IPSC frequency was more marked than the increase in IPSC kinetics. Theoretical considerations suggest that these modifications are sufficient to produce interictal-like activity. In epileptic animals, the ratio of the glutamatergic drive to the overall synaptic drive was not further modified, despite additional changes in synaptic current frequency and kinetics. These results show that the global changes to more glutamatergic and less GABAergic activities in the CA1 region precede the chronic stage of epilepsy, possibly facilitating the occurrence and/or the propagation of interictal activity.     

Pathogenesis of antiphospholipid syndrome: recent insights and emerging concepts

Introduction: Even though our understanding of the antiphospholipid syndrome (APS) has improved tremendously over the last decades, we are still not in a position to replace symptomatic anticoagulation by pathogenesis based causal treatments.

Areas covered: Recent years have provided further insights into pathogenetically relevant mechanisms. These include a differentiation of pathogenic subtypes of antiphospholipid antibodies (aPL), novel mechanisms modulating disease activity, for example, extracellular vesicles and microRNA, and novel players in pathogenesis, for example, neutrophils and neutrophil extracellular traps (NETs).

Expert commentary: It is evident that aPL induce a proinflammatory and procoagulant state and recent data suggest that different aPL species activate different signaling pathways which sometimes converge into a common cellular response. This implies that presence of more than one aPL species may disproportionally increase the risk for the major manifestations of APS, that is, thrombosis and fetal loss. Further delineation of the pathogenic mechanisms will hopefully provide clues to causal rather than symptomatic treatments of APS.     

Localization of endogenous galactoside-binding lectin during morphogenesis ofXenopus laevis

Summary A monoclonal antibody has been produced againstXenopus laevis galactoside-binding neural-creststage lectin. This antibody inhibits lectin-mediated hemagglutination. Using this antibody in conjunction with immunohistochemical techniques, lectin deposition has been studied in embryos and tadpoles at different stages of morphogenesis, from initial neural crest migration, up to the formation of a swimming tadpole. Lectin levels change during development in different regions of the embryo and tadpole, decreasing in migratory cells, and increasing in sites where cells become more adhesive to one another. The results suggest that galactoside-binding lectins may be an important class of cellular adhesion molecules during these stages of development.     

Localization of motoneurons innervating the stylopharyngeus muscle in the cat

Recent investigations of the nucleus ambiguus (NA) have attempted to identify motoneurons associated with muscles of the larynx and pharynx. However, relatively little attention has been directed to the stylopharyngeus muscle, which is important in elevation of the pharynx in swallowing and speech. The present study was designed to identify the specific location of stylopharyngeus motoneurons within the brainstem. Horseradish peroxidase or fluorescent dye was injected into the stylopharyngeus muscle of 12 cats. Retrogradely labeled cells were located ipsilateral in the rostral NA and retrofacial nucleus. This is the first report to definitively localize stylopharyngeus motoneurons.