Increased Voluntary Ethanol Consumption and Changes in Hippocampal Synaptic Plasticity in Isolated C57BL/6J Mice

Social isolation (SI) is a notable model of prolonged mild stress, characterized by multiple neurochemical and behavioral alterations, that appears particularly suitable for studying different aspects of the interplay between stress and ethanol (EtOH) consumption in order to characterize potential molecular mechanisms, including changes in the function of inhibitory GABAergic synapses, underlying such interaction. In C57BL/6J mice, SI is associated with an altered hippocampal concentration of the neuroactive steroids 3α-hydroxy-5α-pregnan-20-one (3α-5α-THP), an increased expression of the α₄ and δ subunit of γ-aminobutyric acid type A receptors (GABA_ARs) in the dentate gyrus (DG), and a parallel enhancement of the stimulatory action of 4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridin-3-ol (THIP) on GABAergic tonic currents recorded in voltage-clamped DG granule cells (DGGCs). In addition, SI in C57BL/6J mice determines an increase in voluntary EtOH consumption and EtOH preference when compared to group-housed (GH) control animals. Furthermore, in hippocampal slices of SI mice we also observed a marked reduction of both cellular excitability and long term potentiation (LTP) in pyramidal neurons of the CA1 hippocampal sub-region, effects that were prevented by the long term treatment of SI mice with the neuroactive steroid precursor progesterone. In this article, we summarize some of our recent findings on the effects of SI in C57BL/6J mice on voluntary EtOH intake, regulation of GABA_ARs gene expression and function and hippocampal long term synaptic plasticity.     

Cdc42 controls the dilation of the exocytotic fusion pore by regulating membrane tension

Membrane fusion underlies multiple processes, including exocytosis of hormones and neurotransmitters. Membrane fusion starts with the formation of a narrow fusion pore. Radial expansion of this pore completes the process and allows fast release of secretory compounds, but this step remains poorly understood. Here we show that inhibiting the expression of the small GTPase Cdc42 or preventing its activation with a dominant negative Cdc42 construct in human neuroendocrine cells impaired the release process by compromising fusion pore enlargement. Consequently the mode of vesicle exocytosis was shifted from full-collapse fusion to kiss-and-run. Remarkably, Cdc42-knockdown cells showed reduced membrane tension, and the artificial increase of membrane tension restored fusion pore enlargement. Moreover, inhibiting the motor protein myosin II by blebbistatin decreased membrane tension, as well as fusion pore dilation. We conclude that membrane tension is the driving force for fusion pore dilation and that Cdc42 is a key regulator of this force.     

Lectin activity of Lentinus edodes

The hemagglutinating activity of submerged mycelium and culture liquid for four strains of Lentinus edodes (Berk.) Sing [L. edodes (Berk.) Pegler] was studied in the search for lectins. The hemagglutinating activity of culture liquid was substantially higher, compared with mycelium. The carbohydrate-binding capacity of the agglutinins was established, and the lectin activity of extracts from mycelia grown on several agar media was elucidated in relation to fruiting. The lectin activity of L. edodes was examined at different morphogenetic steps: mycelium, brown mycelial film, primordium, and fruiting body. Hemagglutination titers at the brown film step were higher than in the mycelium, whereas activity at the primordial and fruiting bodies steps decreased. Lectins seem to be involved in the formation of hyphal aggregates of brown mycelial film.     

An electroporation protocol for efficient DNA transfection in PC12 cells

A wide variety of mammalian cell types is used in gene transfection studies. Establishing transfection methods that enable highly efficient DNA uptake has become increasingly important. PC12 is an established rat pheochromocytoma cell line, which responds to exposure to NGF with cessation of growth, expression of cytoplasmic processes, and differentiation into cells resembling sympathetic neurons. Although PC12 cells represent an important model system to study a variety of neuronal functions, they proved relatively difficult to transfect. We have compared the efficiency of three different chemical transfection reagents (Lipofectamine 2000, Lipofectamine LTX and TransIT-LT1) and of two electroporation systems (Neon and Gene Pulser Xcell) in transiently transfecting undifferentiated PC12 cells. By comparing efficiencies from replicate experiments we proved electroporation (in particular Neon) to be the method of choice. By optimizing different parameters (voltage, pulse width and number of pulses) we reached high efficiency of transfection (90 %) and viability (99 %). We also demonstrated that, upon electroporation, cells are not altered by the transfection and maintain their ability to differentiate.     

A novel stable isotope labelling assisted workflow for improved untargeted LC–HRMS based metabolomics research

Many untargeted LC–ESI–HRMS based metabolomics studies are still hampered by the large proportion of non-biological sample derived signals included in the generated raw data. Here, a novel, powerful stable isotope labelling (SIL)-based metabolomics workflow is presented, which facilitates global metabolome extraction, improved metabolite annotation and metabolome wide internal standardisation (IS). The general concept is exemplified with two different cultivation variants, (1) co-cultivation of the plant pathogenic fungi Fusarium graminearum on non-labelled and highly ¹³C enriched culture medium and (2) experimental cultivation under native conditions and use of globally U-¹³C labelled biological reference samples as exemplified with maize and wheat. Subsequent to LC–HRMS analysis of mixtures of labelled and non-labelled samples, two-dimensional data filtering of SIL specific isotopic patterns is performed to better extract truly biological derived signals together with the corresponding number of carbon atoms of each metabolite ion. Finally, feature pairs are convoluted to feature groups each representing a single metabolite. Moreover, the correction of unequal matrix effects in different sample types and the improvement of relative metabolite quantification with metabolome wide IS are demonstrated for the F. graminearum experiment. Data processing employing the presented workflow revealed about 300 SIL derived feature pairs corresponding to 87–135 metabolites in F. graminearum samples and around 800 feature pairs corresponding to roughly 350 metabolites in wheat samples. SIL assisted IS, by the use of globally U-¹³C labelled biological samples, reduced the median CV value from 7.1 to 3.6 % for technical replicates and from 15.1 to 10.8 % for biological replicates in the respective F. graminearum samples.     

Involvement of the P2X7 Purinergic Receptor in Colonic Motor Dysfunction Associated with Bowel Inflammation in Rats

Background and Purpose

Recent evidence indicates an involvement of P2X7 purinergic receptor (P2X7R) in the fine tuning of immune functions, as well as in driving enteric neuron apoptosis under intestinal inflammation. However, the participation of this receptor in the regulation of enteric neuromuscular functions remains undetermined. This study was aimed at investigating the role of P2X7Rs in the control of colonic motility in experimental colitis.

Experimental Approach

Colitis was induced in rats by 2,4-dinitrobenzenesulfonic acid. P2X7R distribution was examined by immunofluorescence analysis. The effects of A804598 (selective P2X7R antagonist) and BzATP (P2X7R agonist) were tested on contractions of longitudinal smooth muscle evoked by electrical stimulation or by carbachol in the presence of tetrodotoxin.

Key Results

P2X7Rs were predominantly located in myenteric neurons, but, in the presence of colitis, their expression increased in the neuromuscular layer. In normal preparations, A804598 elicited a negligible increase in electrically induced contractions, while a significant enhancement was recorded in inflamed tissues. In the presence of N^ω-propyl-L-arginine (NPA, neuronal nitric oxide synthase inhibitor) the A804598 effects were lost. P2X7R stimulation with BzATP did not significantly affect electrical-induced contractions in normal colon, while a marked reduction was recorded under inflammation. The inhibitory effect of BzATP was antagonized by A804598, and it was also markedly blunted by NPA. Both P2X7R ligands did not affect carbachol-induced contractions.

Conclusions and Implications

The purinergic system contributes to functional neuromuscular changes associated with bowel inflammation via P2X7Rs, which modulate the activity of excitatory cholinergic nerves through a facilitatory control on inhibitory nitrergic pathways.     

Pollen wall ontogeny in <Emphasis Type="Italic">Polemonium caeruleum</Emphasis> (Polemoniaceae) and suggested underlying mechanisms of development

By a detailed ontogenetic study of Polemonium caeruleum pollen, tracing each stage of development at high TEM resolution, we aim to understand the establishment of the pollen wall and to unravel the mechanisms underlying sporoderm development. The main steps of exine ontogeny in Polemonium caeruleum, observed in the microspore periplasmic space, are spherical units, gradually transforming into columns, then to rod-like units (procolumellae), the appearance of the initial tectum, growth of columellae in height and tectum in thickness and initial sporopollenin accumulation on them, the appearance of the endexine lamellae and of dark-contrasted particles on the tectum, the appearance of a sponge-like layer and of the intine in aperture sites, the appearance of the foot layer on the base of the sponge-like layer and of spinules on the tectum, and massive sporopollenin accumulation. This sequence of developmental events fits well to the sequence of self-assembling micellar mesophases. This gives (together with earlier findings and experimental exine simulations) strong evidence that genome and self-assembly probably share control of exine formation. It is highly probable that self-assembly is an intrinsic instrument of evolution.     

Reproducible reference frame for in vitro testing of the human vertebrae

Definition of an anatomical reference frame is necessary for in vitro biomechanical testing. Nevertheless, there is neither a clear recommendation, nor consensus in the literature concerning an anatomical reference frame for in vitro testing of the human vertebrae. The scope of this work is to define a reference frame for the human vertebrae for in vitro applications. The proposed anatomical reference frame relies on alignment of well-defined points on the endplates, and on two landmarks on the posterior wall. The repeatability of the proposed alignment procedure has been tested in vitro by 5 operators, on 7 specimens. Furthermore, the feasibility and repeatability of the proposed procedure was assessed in silico, using CT-scans of the same specimens.