Role of astrocytic connexins in health and disease

Despite of the growing number of studies, the expression pattern as well as the exact functions of astrocytic connexins are not yet fully elucidated. In this study I analyzed the expression pattern and functions of the two major astrocytic connexins, Cx30 and Cx43, under normal conditions and in a mouse model for temporal lobe epilepsy (TLE). In the first part of my work I characterized novel conditional knock out mice, expressing ECFP instead of Cx43 after Cre-mediated recombination. Utilizing this mouse, I demonstrated dual reporter approaches to i) simultaneously examine astrocyte subpopulations expressing different connexins, ii) identify compensatory upregulation within gene families and iii) quantify Cre-mediated deletion at the allelic level. In the second part of my work, I re-evaluated the expression of Cx30 in different brain regions. I analyzed the reporter gene expression of Cx30 knockout mice. Utilizing a fate mapping approach, I showed that the reporter gene expression does not reflect properly the Cx30 expression in the hippocampus. Using a fate mapping approach, I also demonstrated that Cx30 is expressed at similar levels compared to Cx43 in most brain regions. In addition, utilizing the same fate mapping approach, I showed that radial glia like cells express mostly Cx43, indicating that Cx43 is the most important connexin for adult neurogenesis. In the third part of my work, I analyzed the influence of Cx30 and Cx43 on morphological changes in brain of mice subjected to a novel mouse TLE model (intracortical kainate injection). In mice lacking both Cx43 and Cx30, I observed less pronounced morphological changes in the hippocampus. I also established methods for quality control of the trangenic mice used in this study. My study adds to a better understanding of expression pattern of astrocytic connexines in the brain, and its role in the epilepsy

Measurement of ϒ production in pp collisions at √s = 2.76 TeV

The production of ϒ(1S), ϒ(2S) and ϒ(3S) mesons decaying into the dimuon final state is studied with the LHCb detector using a data sample corresponding to an integrated luminosity of 3.3 pb−1 collected in proton–proton collisions at a centre-of-mass energy of √s = 2.76 TeV. The differential production cross-sections times dimuon branching fractions are measured as functions of the ϒ transverse momentum and rapidity, over the ranges pT < 15 GeV/c and 2.0 < y < 4.5. The total cross-sections in this kinematic region, assuming unpolarised production, are measured to be σ (pp → ϒ(1S)X) × B ϒ(1S)→μ+μ− = 1.111 ± 0.043 ± 0.044 nb, σ (pp → ϒ(2S)X) × B ϒ(2S)→μ+μ− = 0.264 ± 0.023 ± 0.011 nb, σ (pp → ϒ(3S)X) × B ϒ(3S)→μ+μ− = 0.159 ± 0.020 ± 0.007 nb, where the first uncertainty is statistical and the second systematic

Dysbalance of Astrocyte Calcium under Hyperammonemic Conditions

Increased brain ammonium (NH4(+)/NH3) plays a central role in the manifestation of hepatic encephalopathy (HE), a complex syndrome associated with neurological and psychiatric alterations, which is primarily a disorder of astrocytes. Here, we analysed the influence of NH4(+)/NH3 on the calcium concentration of astrocytes in situ and studied the underlying mechanisms of NH4(+)/NH3-evoked calcium changes, employing fluorescence imaging with Fura-2 in acute tissue slices derived from different regions of the mouse brain. In the hippocampal stratum radiatum, perfusion with 5 mM NH4(+)/NH3 for 30 minutes caused a transient calcium increase in about 40% of astrocytes lasting about 10 minutes. Furthermore, the vast majority of astrocytes (∼ 90%) experienced a persistent calcium increase by ∼ 50 nM. This persistent increase was already evoked at concentrations of 1-2 mM NH4(+)/NH3, developed within 10-20 minutes and was maintained as long as the NH4(+)/NH3 was present. Qualitatively similar changes were observed in astrocytes of different neocortical regions as well as in cerebellar Bergmann glia. Inhibition of glutamine synthetase resulted in significantly larger calcium increases in response to NH4(+)/NH3, indicating that glutamine accumulation was not a primary cause. Calcium increases were not mimicked by changes in intracellular pH. Pharmacological inhibition of voltage-gated sodium channels, sodium-potassium-chloride-cotransporters (NKCC), the reverse mode of sodium/calcium exchange (NCX), AMPA- or mGluR5-receptors did not dampen NH4(+)/NH3-induced calcium increases. They were, however, significantly reduced by inhibition of NMDA receptors and depletion of intracellular calcium stores. Taken together, our measurements show that sustained exposure to NH4(+)/NH3 causes a sustained increase in intracellular calcium in astrocytes in situ, which is partly dependent on NMDA receptor activation and on release of calcium from intracellular stores. Our study furthermore suggests that dysbalance of astrocyte calcium homeostasis under hyperammonemic conditions is a widespread phenomenon, which might contribute to the disturbance of neurotransmission during HE

NH₄⁺-induced changes in neocortex and cerebellum.

<p><b>(A)</b> Calcium changes evoked by bath perfusion with 5 mM NH₄⁺/NH₃ for 30 minutes (indicated by bar) in two different SR101-positive astrocytes of the sensory cortex. Upper trace: biphasic response, consisting of a transient elevation followed by a plateau phase. Lower trace: monophasic response with a plateau phase only. <b>(B)</b> Calcium changes evoked by bath perfusion with 5 mM NH₄⁺/NH₃ for 30 minutes (indicated by bar) in two different Bergmann glial cells of the cerebellar cortex. Upper trace: biphasic response, consisting of a transient elevation followed by a plateau phase. Lower trace: monophasic response with a plateau phase only.</p

Involvement of glutamate receptors in NH₄⁺/NH₃-induced calcium changes.

<p><b>(A)</b> Influence of the NMDA-receptor blocker DL-AP5 (100 µM, indicated by bar) on NH₄⁺/NH₃-induced calcium changes in a biphasic (upper trace) and a monophasic (lower trace) hippocampal astrocyte. The grey bars indicate the average amplitude of the sustained calcium increase evoked under control conditions in the absence of DLAP5 (∼50 nM). <b>(B)</b> Histogram showing the mean peak amplitude ± S. E. M. of sustained calcium changes in response to NH₄⁺/NH₃ in the control, and in the presence of the glutamate receptors blocker CNQX, DLAP5 (AP5), DLAP5 in the presence of TTX, or MPEP. The number of cells is given within the bars. AP5 and AP5/TTX reduce the amplitude of calcium increases significantly as compared to the control (***: p<0.001; *: p<0.05).</p

Interrelationship between NH₄⁺-induced changes in calcium and pH.

<p><b>(A)</b> Changes in intracellular pH (pH_i) evoked by bath perfusion with 5 mM NH₄⁺/NH₃ for 30 minutes (indicated by bar) in a SR101-positive hippocampal astrocyte. <b>(B)</b> Changes in a hippocampal astrocyte evoked by perfusion with the weak base trimethylamine (TMA, 10 mM) for 30 minutes (indicated by bar). Note the pronounced alkalinization, which is followed by an acidification upon removal of TMA. <b>(C)</b> Trace showing intracellular calcium in a hippocampal astrocyte and the influence of perfusion with TMA (10 mM). Note that TMA does not evoke any changes in astrocyte calcium.</p

Dependence of astrocyte calcium changes on NH₄⁺/NH₃-concentrations.

<p><b>(A)</b> Calcium changes in an hippocampal astrocyte evoked by stepwise increases in the NH₄⁺/NH₃ concentration by 0.5 mM for 2 minutes, starting from nominally 0 mM up to a final concentration of 5 mM which was then maintained for another 15 minutes. <b>(B)</b> Influence of methioninesulfoximine (MSO, 100 µM) on NH₄⁺/NH₃-induced calcium changes in a hippocampal astrocyte. The grey bar indicates the average amplitude of the sustained calcium increase evoked under control conditions in the absence of MSO (∼50 nM). <b>(C)</b> Histogram showing the mean peak amplitude ± S. E. M. of sustained calcium changes in response to 1, 2 and 5 mM NH₄⁺/NH₃, after stepwise increases from 0 to 5 mM NH₄⁺/NH₃ and in the presence of MSO and 5 mM NH₄⁺/NH₃. The number of cells is given within the bars. Peak amplitudes of calcium changes did not differ between the different concentrations used. They were, however, significantly larger in the presence of MSO as compared to all other conditions (*: p<0.05; **: p<0.01).</p

Involvement of calcium influx and intracellular stores in NH₄⁺/NH₃-induced calcium changes.

<p><b>(A)</b> Influence of combined removal of extracellular calcium with the NMDA-receptor blocker DLAP5 (“0Ca²⁺/100 µM DLAP5”; indicated by bar) on NH₄⁺/NH₃-induced calcium changes in a hippocampal astrocyte. Note the decrease in calcium upon removal of calcium. <b>(B)</b> Influence of the SERCA blocker CPA (10 µM, indicated by bar) on NH₄⁺/NH₃-induced calcium changes in a hippocampal astrocyte. Note the biphasic calcium elevation induced at the onset of CPA perfusion. <b>(A)</b>, <b>(B)</b> The grey bars indicate the average amplitude of the sustained calcium increase evoked under control conditions in the absence of blockers (∼50 nM) <b>(C)</b> Histogram showing the mean peak amplitude ± S. E. M. of sustained calcium changes in response to NH₄⁺/NH₃ in the control, in the nominal absence of extracellular calcium, in the absence of extracellular calcium combined DLAP5, in the presence of CPA and in the combined presence of CPA and DLAP5. The number of cells is given within the bars; all manipulations result in significantly smaller changes in calcium as compared to the control (***: p<0.001; **: p<0.01; *: p<0.05).</p

Germ-line recombination activity of the widely used hGFAP-Cre and nestin-Cre transgenes.

Herein we demonstrate with PCR, immunodetection and reporter gene approaches that the widely used human Glial Fibrillary Acidic Protein (hGFAP)-Cre transgene exhibits spontaneous germ-line recombination activity in leading to deletion in brain, heart and tail tissue with high frequency. The ectopic activity of hGFAP-Cre requires a rigorous control. We likewise observed that a second widely used nestin-Cre transgene shows germ-line deletion. Here we describe procedures to identify mice with germ-line recombination mediated by the hGFAP-Cre and nestin-Cre transgenes. Such control is essential to avoid pleiotropic effects due to germ-line deletion of loxP-flanked target genes and to maintain the CNS-restricted deletion status in transgenic mouse colonies

Immunoblot analysis of hippocampal lysates for Cre and Cx43 protein indicates loss of Cx43 expression in the absence of Cre protein.

<p>In Cx43^fl/del mice (green box), immunoreactivity for Cx43 is reduced by about 50% compared to Cx43^fl/fl mice. Immunoreactivity for the full length Cx43 is completely lost in Cx43^K258Stop/del mice (red box). Please note absence of Cre immunoreactivity in the Cx43^fl/del and Cx43^K258Stop/del lanes, consistent with negative PCR results for hGFAP-Cre and internal Cre PCRs. Upper row: Cx43 immunoreactivity at 43 kDa (full length protein, Cx43) with an antibody directed to the C-terminus. Second row: Cx43 immunoreactivity at 28 kDa (truncated protein, Cx43K258stop) with an antibody directed to the N-terminus. Third row: Immunoreactivity for the Cre recombinase (Cre). Fourth row: Tubulin loading control. WT: Cx43^+/+. kDa: Kilodalton.</p