CMS physics technical design report : Addendum on high density QCD with heavy ions

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To Break or to Brake Neuronal Network Accelerated by Ammonium Ions?

The aim of present study was to investigate the effects of ammonium ions on in vitro neuronal network activity and to search alternative methods of acute ammonia neurotoxicity prevention.Rat hippocampal neuronal and astrocytes co-cultures in vitro, fluorescent microscopy and perforated patch clamp were used to monitor the changes in intracellular Ca2+- and membrane potential produced by ammonium ions and various modulators in the cells implicated in neural networks.Low concentrations of NH4Cl (0.1-4 mM) produce short temporal effects on network activity. Application of 5-8 mM NH4Cl: invariably transforms diverse network firing regimen to identical burst patterns, characterized by substantial neuronal membrane depolarization at plateau phase of potential and high-amplitude Ca2+-oscillations; raises frequency and average for period of oscillations Ca2+-level in all cells implicated in network; results in the appearance of group of «run out» cells with high intracellular Ca2+ and steadily diminished amplitudes of oscillations; increases astrocyte Ca2+-signalling, characterized by the appearance of groups of cells with increased intracellular Ca2+-level and/or chaotic Ca2+-oscillations. Accelerated network activity may be suppressed by the blockade of NMDA or AMPA/kainate-receptors or by overactivation of AMPA/kainite-receptors. Ammonia still activate neuronal firing in the presence of GABA(A) receptors antagonist bicuculline, indicating that «disinhibition phenomenon» is not implicated in the mechanisms of networks acceleration. Network activity may also be slowed down by glycine, agonists of metabotropic inhibitory receptors, betaine, L-carnitine, L-arginine, etc.Obtained results demonstrate that ammonium ions accelerate neuronal networks firing, implicating ionotropic glutamate receptors, having preserved the activities of group of inhibitory ionotropic and metabotropic receptors. This may mean, that ammonia neurotoxicity might be prevented by the activation of various inhibitory receptors (i.e. by the reinforcement of negative feedback control), instead of application of various enzyme inhibitors and receptor antagonists (breaking of neural, metabolic and signaling systems)

Transformation of simple and complex intracellular Ca²⁺-oscillations into high-amplitude impulse-shaped Ca²⁺-oscillations by NH₄Cl or bicuculline.

<p>Neuronal cultures 12 DIV. Resting calcium level is outlined by dot-dashed lines. Calcium increment over resting level (VCi = ΔCa (a.u.)/min) is indicated on the Figures as VCi. All other abbreviations as on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134145#pone.0134145.g001" target="_blank">Fig 1</a>. Total number of neuronal cells in networks are: N = 116, 132, 98, 110 for Fig A, B, C and D, correspondingly. <b>(A</b>–<b>C)</b> NH₄Cl induces high-amplitude Ca²⁺-oscillations in representative cells. 200 nM of L-glutamate was added before application of NH₄Cl. <b>(C)</b> The experiment was performed in the presence of 10 μM L-NAME and 200 nM of L-glutamate. <b>(D)</b> 10 μM of bicuculline evokes high-amplitude Ca²⁺-oscillations in spontaneously firing cell. Only parts of total records are presented on Fig B and C. Initial parts were omitted for simplicity.</p

Inhibitory effect of betaine on the activation of networks by ammonium ions and its dependence on the operation of GABA(A)-receptors.

<p>Cultures 14 DIV. The records of representative cells. All other abbreviations and descriptions as on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134145#pone.0134145.g002" target="_blank">Fig 2</a>. <b>(A)</b> Suppression of Ca²⁺-oscillations induced by 8mM NH₄Cl after the application 10 mM of betaine. N = 136. <b>(B</b>) Disappearance of suppressive effect of betaine in the presence of GABA(A)-receptors antagonist bicuculline (10 μM). N = 124. 200 nM of L-glutamate was added before application of NH₄Cl on Fig B.</p

Suppressive and modulating effects of L-arginine, L-carnitine and acetyl-L-carnitine on neuronal networks activated by ammonium ions.

<p>Cultures 12–18 DIV. The records of representative cells (more than 90% of cells in culture) are presented. All other abbreviations and descriptions as on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134145#pone.0134145.g002" target="_blank">Fig 2</a>. <b>(A, B)</b> Modulating (A) and suppressive (B) effects of L-arginine (10 mM). Typical responses of cells in few (Fig A; 20%) and in most (80%; Fig B) of cultures studied. n = 10. Culture 12 DIV. N = 89 (for Fig A). N = 106 (for Fig B). <b>(C, D)</b> Typical suppressant effect of L-carnitine (10 mM; Fig C) and modulatory effect of acetyl-L-carnitine (10 mM; Fig D) in most of cultures studied (75 and 80%, of cultures. n = 4 and n = 5 correspondingly). Culture 16 DIV. N = 111 (for Fig C). Culture 18 DIV. N = 126 (for Fig D). 200 nM of L-glutamate was added before application of 8 mM NH₄Cl on Fig B, C, D.</p

Comparative simultaneous recordings of Ca²⁺-oscillations in representative neuronal cell (A) and calcium signaling in two types of astrocytes (B) in network activated by 6 mM NH₄Cl.

<p>Neuronal culture 14 DIV. Part of the experiment presented on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134145#pone.0134145.g004" target="_blank">Fig 4</a>. All conditions as at <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134145#pone.0134145.g004" target="_blank">Fig 4</a>. <b>(A)</b> Recording of Ca²⁺ oscillations in representative neuronal cell (95% of cells) after application of NH₄Cl. This is the repeat of the recording presented on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134145#pone.0134145.g004" target="_blank">Fig 4A</a>. <b>(B)</b> Recordings of two types of responses of representative astrocytes are shown. NH₄Cl slightly increases astrocyte Ca²⁺_i level with generation of solitary Ca²⁺ spikes in 44 of 67 cells (black line) and induce chaotic Ca²⁺ oscillations in 23 of 67 cells (gray line).</p

Simultaneous recordings of membrane potential and of Ca²⁺-oscillations in «run out» cell and representative cell in network activated by 5 mM NH₄Cl.

<p>Neuronal culture 16 DIV. Total number of neuronal cells involved into network is 106. Gaps in the traces represent pauses in data recordings. Here are presented only parts of 3 records. Initial parts were omitted for simplicity. <b>(A)</b> Recording of Ca²⁺-oscillations in «run out» cell. <b>(B)</b> Recording of membrane potential in «run out» cell. <b>(C)</b> Recording of Ca²⁺-oscillations in representative cell (one of 95% cells monitored in network).</p

Ammonium chloride effects on neuronal network are concentration dependent.

<p>Here and later on calcium responses are measured by Fura-2 ratio. Records characterize individual neurons (black lines) and astrocytes (grey lines), i.e. representative cells of 90–95% cells implicated in the network. Thick horizontal black lines mark the periods of application of ammonium chloride (NH₄Cl, 0.1–4 mM), L-glutamate (200 nM) and KCl (35 mM). <b>(A)</b> Application of 0.1 mM NH₄Cl does not alter Ca²⁺ signals registered in representative cells: neurone (black line) and astrocyte (grey line). Neuronal culture 12 DIV. Number of monitored neurons in network N = 84 and number of astrocytes N₁ = 47. Here is presented one of 12 experiments (n = 12). <b>(B)</b> Application of 1 mM NH₄Cl induces one burst of high-amplitude Ca²⁺ oscillations in representative neuronal cell (black line) and does not alter significantly Ca²⁺ level in representative astrocyte (grey line). Neuronal culture 12 DIV. Number of neurons in network N = 96 and number of monitored astrocytes N₁ = 52. Here is presented 1 of 3 experiments with evoked Ca²⁺ signal. Total number of experiments n = 20. <b>(C)</b> Application of 2 mM NH₄Cl induces one burst of high-amplitude Ca²⁺ oscillations in representative neuronal cell (black line) wich is accompanied by some temporary elevation in astrocytic Ca²⁺-signal (grey line). Neuronal culture 15 DIV. Total number of neurons involved into network N = 102. Number of monitored astrocytes N₁ = 43. Here is presented 1 of 2 experiments with evoked Ca²⁺-burst. Total number of experiments n = 10. <b>(D)</b> Application of 3 mM NH₄Cl induces one burst of high-amplitude Ca²⁺ oscillations in representative neuronal cell (black line) with the rise in Ca²⁺ level in after burst period. Neuronal culture 14 DIV. Total number of neurons involved into network N = 91. Number of monitored astrocytes N₁ = 47. Here is presented 1 of 3 experiments with evoked Ca²⁺-burst. Total number of experiments n = 12. <b>(E)</b> Induction of sustained Ca²⁺-oscillations in neuronal network by 4 mM NH₄Cl. Record of representative neuron is presented. Neuronal culture 5 DIV. Total number of neurons in network N = 84. Here is presented 1 of 6 experiments with stable high-amplitude Ca²⁺-oscillations. Total number of experiments n = 10. 200 nM of L-glutamate was added before application of NH₄Cl. <b>(F)</b> The record of Ca²⁺-signalling in astrocyte evoked by 3 mM NH₄Cl. It corresponds to the experiment presented on Fig D. Application of 3 mM NH₄Cl induces immediate rise of Ca²⁺ level to new steady state in representative astrocyte (grey line). Note the significant differences in the responses of neurons (Fig B) and astrocytes to depolarizing action of 35 mM KCl. Neuronal culture 14 DIV. Total number of neurons N = 91. Number of monitored astrocytes N₁ = 47. Inserted black bars indicate the average amplitudes ± SD of intracellular Ca²⁺ level in 47 astrocytes recorded at time-points indicated. *<i>P</i><0.05 is given for difference between both values.</p

Synergistic action of ammonium ions and NMDA on changes in calcium concentrations in immature neuronal cells.

<p>Neuronal cultures 5 and 7 DIV. All other abbreviations and descriptions as on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134145#pone.0134145.g001" target="_blank">Fig 1</a>. Gaps in the traces represent pauses in data recordings. <b>(A)</b> Combined synergistic action of NH₄Cl (4 mM) and NMDA (10 μM) on cellular Ca²⁺ level, both of each separately cannot evoke Ca²⁺ signal and activate the cell. Culture 5 DIV. N = 66. <b>(B)</b> Neuronal Ca²⁺ responses to separate and combined action of NMDA (20 μM) and KCl (5 mM). Culture 7 DIV. N = 72.</p