Abstract
PurposeThe 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.MethodsRat 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.ResultsLow 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.ConclusionsObtained 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).
Highlights
It has long been known that the excess of ammoniacan lead to lethargy, convulsions, ataxia and coma in patients with hepatic encephalopathy (HE) [1, 2]
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 Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-receptors or by overactivation of AMPA/kainite-receptors
Summary
It has long been known that the excess of ammonia (sum of NH3 and NH4+)can lead to lethargy, convulsions, ataxia and coma in patients with hepatic encephalopathy (HE) [1, 2]. P. injections of lethal doses of ammonium acetate or NH4Cl may result in initial agitation and drowsiness, followed by clonic and tonic seizures, coma and animal death within 10–20 min [3,4,5]. «Ammonia lowering strategies» are based on the suppression of ammonia production by gut flora (lactulose and antibiotics) and on the activation of ammonia consuming reactions (urea cycle and glutamine synthetase). These strategies represent core elements in the existing options of HE treatment during last fifty years [10,11,12,13,14]. Compositions of L-ornithine-L-aspartate, L-ornithine-α-ketoglutarate or L-ornithine-phenylacetate, etc. are often used to activate urea cycle and glutamine synthetase and to trap ammonia and glutamine [11,12,13,14,15]
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