Biomedical subjects

Bernhard U Keller

Publications and source records attributed to Bernhard U Keller.

7 recordsLinked to original sources

PubMed2005

Mitochondrial Ca2+ buffering in hypoglossal motoneurons from mouse.

A variety of studies demonstrated a crucial role of mitochondria for clearance of Ca2+ loads in motoneurons. However, previous reports rarely addressed the potential influence of cell dialysis during patch-clamp recordings or temperature on mitochondrial processes. We therefore developed a protocol allowing investigation of Ca2+ dynamics in "undisturbed" AM-ester loaded hypoglossal motoneurons in a slice preparation. By comparing our findings to previous results, we argue against a significant disturbance of mitochondrial buffering by cell dialysis. By varying bath temperatures between 19 and 32 degrees C, we show that temperature alters the rate of mitochondrial uptake but not the relative contribution to maintenance of Ca2+ homeostasis. The results further indicate that mitochondria in hypoglossal motoneurons participate in intracellular Ca2+ regulation at concentrations much lower than has been generally observed for other neurons or neuroendocrine cells. Taken together, our findings further support the important role of mitochondria as regulators of Ca2+ homeostasis in motoneurons.

Animals↗

PubMed2005

Ca2+, mitochondria and selective motoneuron vulnerability: implications for ALS.

Motoneurons are selectively damaged in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. Although the underlying mechanisms are not completely understood, increasing evidence indicates that motoneurons are particularly sensitive to disruption of mitochondria and Ca(2+)-dependent signalling cascades. Comparison of ALS-vulnerable and ALS-resistant neurons identified low Ca(2+)-buffering capacity and a strong impact of mitochondrial signal cascades as important risk factors. Under physiological conditions, weak Ca(2+) buffers are valuable because they facilitate rapid relaxation times of Ca(2+) transients in motoneurons during high-frequency rhythmic activity. However, under pathological conditions, weak Ca(2+) buffers are potentially dangerous because they accelerate a vicious circle of mitochondrial disruption, Ca(2+) disregulation and excitotoxic cell damage.

Amyotrophic Lateral Sclerosis↗

PubMed2004

Functional analysis of glutamate transporters in excitatory synaptic transmission of GLAST1 and GLAST1/EAAC1 deficient mice.

The high affinity, Na(+)-dependent, electrogenic glial L-glutamate transporters GLAST1 and GLT1, and two neuronal EAAC1 and EAAT4, regulate the neurotransmitter concentration in excitatory synapses of the central nervous system. We dissected the function of the individual transporters in the monogenic null allelic mouse lines, glast1(-/-) and eaac1(-/-), and the derived double mutant glast(-/-)eaac1(-/-). Unexpectedly, the biochemical analysis and the behavioral phenotypes of these null allelic mouse lines were inconspicuous. Inhibition studies of the Na(+)-dependent glutamate transport by plasma membrane vesicles and by isolated astrocytes of wt and glast1(-/-) mouse brains indicated the pivotal compensatory role of GLT1 in the absence particularly of GLAST1 and GLAST1 and EAAC1 mutant mice. In electrophysiological studies, the decay rate of excitatory postsynaptic currents (EPSCs) of Purkinje cells (PC) after selective activation of parallel and climbing fibers proved to be similar in wt and eaac1(-/-), but was significantly prolonged in glast1(-/-) PCs. Bath application of the glutamate uptake blocker SYM2081 prolonged EPSC decay profiles in both wt and double mutant glast1(-/-)eaac1(-/-) PCs by 286% and 229%, respectively, indicating a prominent role of compensatory glutamate transport in shaping glast1(-/-)eaac1(-/-) EPSCs.

Amino Acid Transport System X-AG↗

PubMed2004

Serotonergic modulation of intracellular calcium dynamics in neonatal hypoglossal motoneurons from mouse.

(1) Serotonin (5HT)-mediated calcium signaling was investigated in hypoglossal motoneurons (HGMs) in brain stem slices of neonatal mice. Electrical activity and associated calcium signaling were studied by simultaneous patch clamp recordings and high resolution calcium imaging. (2) Bath application of 5HT (5-50 microM) depolarized membrane potential of HGMs and generated action potential discharges that were accompanied by elevations in intracellular calcium concentrations ([Ca2+]i) in the soma and dendrites. Current-evoked bursts of action potentials were more intense in the presence of 5HT; however, the corresponding calcium signals were reduced. (3) The 5HT2 receptor agonist alpha-Methyl-5HT (25, 50 microM) had effects on membrane potential, discharge properties and [Ca]i that were identical to those observed for 5HT, whereas the 5HT3 receptor agonist 1-(m-chlorophenyl) biguanide (50 microM) had no effect on membrane properties or intracellular calcium levels. (4) 8-OHDPAT (25, 50 microM), a 5HT1A receptor agonist, was without effect on steady-state membrane potential or basal [Ca]i. Similar to 5HT and alpha-Methyl-5HT, 8-OHDPAT depressed stimulus-evoked calcium transients in current and voltage clamp mode. (5) Our results suggest that calcium profiles in hypoglossal motoneurons are differentially regulated by 5HT1A and 5HT2 receptors. Activation of 5HT1A receptors primarily reduced voltage-activated Ca2+ signals without a significant impact on basal [Ca]i. In contrast, activation of 5HT2 receptors initiated a net inward current followed by membrane depolarization, where the resulting pattern of action potential discharges represents the essential determinant of global elevations in [Ca2+]i. Taken together, our results therefore identify 5HT-dependent signal pathways as a versatile tool to modulate hypoglossal motoneuron excitability under various physiological and pathophysiological conditions.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed2003

Impact of mitochondrial inhibition on excitability and cytosolic Ca2+ levels in brainstem motoneurones from mouse.

Motoneurones (MNs) are particularly affected by the inhibition of mitochondrial metabolism, which has been linked to their selective vulnerability during pathophysiological states like hypoxia and amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. To elucidate underlying events, we used sodium cyanide (CN) as a pharmacological inhibitor of complex IV of the mitochondrial respiratory chain ('chemical hypoxia') and investigated the cellular response in vulnerable and resistant neurone types. Bath application of 2 mm CN activated TTX-insensitive Na+ conductances in vulnerable hypoglossal MNs, which depolarized these MNs by 10.2 +/- 1.1 mV and increased their action potential activity. This response was mimicked by sodium azide (2 mm) and largely prevented by preincubation with the antioxidants ascorbic acid (1 mm) and Trolox (750 microm), indicating an involvement of reactive oxygen species (ROS) in the activation mechanism. CN also elevated cytosolic [Ca2+] levels through (i) Ca2+ release from mitochondria-controlled stores, (ii) significant retardation of cytosolic Ca2+ clearance rates, even when cytosolic ATP levels were held constant during whole-cell recording, and (iii) secondary Ca2+ influx during elevated firing rates. Blocking mitochondrial ATP production additionally raised cytosolic Ca2+ levels and prolonged recovery of Ca2+ transients with a delay of 5-6 min. Comparative studies on hypoglossal MNs, facial MNs and dorsal vagal neurones suggested that CN responses were dominated by the activation of K+ conductances in resistant neurones, thus reducing excitability during mitochondrial inhibition. In summary, our observations therefore support a model where selective MN vulnerability results from a synergistic accumulation of risk factors, including low cytosolic Ca2+ buffering, strong mitochondrial impact on [Ca2+]i, and a mitochondria-controlled increase in electrical excitability during metabolic disturbances.

Action Potentials↗

PubMed2003

Spatial profiles of store-dependent calcium release in motoneurones of the nucleus hypoglossus from newborn mouse.

Hypoglossal motoneurones (HMN) are selectively damaged in both human amyotrophic lateral sclerosis (ALS) and corresponding mouse models of this neurodegenerative disease, a process which has been linked to their low endogenous Ca2+ buffering capacity and an exceptional vulnerability to Ca2+-mediated excitotoxic events. In this report, we investigated local Ca2+ profiles in low buffered HMNs by utilizing multiphoton microscopy, CCD imaging and patch clamp recordings in slice preparations. Bath application of caffeine induced highly localized Ca2+ release events, which displayed an initial peak followed by a slow 'shoulder' lasting several seconds. Peak amplitudes were paralleled by Ca2+-activated, apamin-sensitive K+ currents (IKCa), demonstrating a functional link between Ca2+ stores and HMN excitability. The potential involvement of mitochondria was investigated by bath application of CCCP, which collapses the electrochemical potential across the inner mitochondrial membrane. CCCP reduced peak amplitudes of caffeine responses and consequently IKCa, indicating that functionally intact mitochondria were critical for store-dependent modulation of HMN excitability. Taken together, our results indicate localized Ca2+ release profiles in HMNs, where low buffering capacities enhance the role of Ca2+-regulating organelles as local determinants of [Ca2+]i. This might expose HMN to exceptional risks during pathophysiological organelle disruptions and other ALS-related, cellular disturbances.

Amyotrophic Lateral Sclerosis↗

PubMed2002

Neurotoxicity of pneumolysin, a major pneumococcal virulence factor, involves calcium influx and depends on activation of p38 mitogen-activated protein kinase.

Neuronal injury in bacterial meningitis is caused by the interplay of host inflammatory responses and direct bacterial toxicity. We investigated the mechanisms by which pneumolysin, a cytosolic pneumococcal protein, induces damage to neurons. The toxicity after exposure of human SH-SY5Y neuroblastoma cells and hippocampal organotypic cultures to pneumolysin was time- and dose-dependent. Pneumolysin led to a strong calcium influx apparently mediated by pores on the cell membrane formed by the toxin itself and not by voltage-gated calcium channels. Buffering of intracellular calcium with BAPTA-AM [1, 2-bis (o-aminophenoxy) ethane N, N, N', N'-tetraacetic acid tetra(acetomethoxyl) ester] improved survival of neuronal cells following challenge with pneumolysin. Western blotting revealed increased phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) as early as 30 min after challenge with pneumolysin. SB 203580, a potent and selective inhibitor of p38 MAPK, rescued human neuronal cells from pneumolysin-induced death. Inhibition of the mitochondrial permeability transition pore using bongkrekate and caspase inhibition also improved survival following challenge with the toxin. Modulation of cell death pathways activated by pneumolysin may influence the outcome of pneumococcal meningitis.

Animals↗