Biomedical subjects

Sebastian Schuchmann

Publications and source records attributed to Sebastian Schuchmann.

14 recordsLinked to original sources

PubMed2006

Experimental febrile seizures are precipitated by a hyperthermia-induced respiratory alkalosis.

Febrile seizures are frequent during early childhood, and prolonged (complex) febrile seizures are associated with an increased susceptibility to temporal lobe epilepsy. The pathophysiological consequences of febrile seizures have been extensively studied in rat pups exposed to hyperthermia. The mechanisms that trigger these seizures are unknown, however. A rise in brain pH is known to enhance neuronal excitability. Here we show that hyperthermia causes respiratory alkalosis in the immature brain, with a threshold of 0.2-0.3 pH units for seizure induction. Suppressing alkalosis with 5% ambient CO2 abolished seizures within 20 s. CO2 also prevented two long-term effects of hyperthermic seizures in the hippocampus: the upregulation of the I(h) current and the upregulation of CB1 receptor expression. The effects of hyperthermia were closely mimicked by intraperitoneal injection of bicarbonate. Our work indicates a mechanism for triggering hyperthermic seizures and suggests new strategies in the research and therapy of fever-related epileptic syndromes.

Alkalosis, Respiratory↗

PubMed2006

The cation-chloride cotransporter NKCC1 promotes sharp waves in the neonatal rat hippocampus.

Earlier studies indicate a crucial role for the interconnected network of intrinsically bursting CA3 pyramidal neurons in the generation of in vivo hippocampal sharp waves (SPWs) and their proposed neonatal in vitro counterparts, the giant depolarizing potentials (GDPs). While mechanisms involving ligand- and voltage-gated channels have received lots of attention in the generation of CA3 network events in the immature hippocampus, the contribution of ion-transport mechanisms has not been extensively studied. Here, we show that bumetanide, a selective inhibitor of neuronal Cl- uptake mediated by the Na+-K+-2Cl- cotransporter isoform 1 (NKCC1), completely and reversibly blocks SPWs in the neonate (postnatal days 7-9) rat hippocampus in vivo, an action also seen on GDPs in slices (postnatal days 1-8). These findings strengthen the view that GDPs and early SPWs are homologous events. Gramicidin-perforated patch recordings indicated that NKCC1 accounts for a large ( approximately 10 mV) depolarizing driving force for the GABAA current in the immature CA3 pyramids. Consistent with a reduction in the depolarization mediated by endogenous GABAA-receptor activation, bumetanide inhibited the spontaneous bursts of individual neonatal CA3 pyramids, but it slightly increased the interneuronal activity as seen in the frequency of spontaneous GABAergic currents. An inhibitory effect of bumetanide was seen on the in vitro population events in the absence of synaptic GABAA receptor-mediated transmission, provided that a tonic GABAA receptor-mediated current was present. Our work indicates that NKCC1 expressed in CA3 pyramidal neurons promotes network activity in the developing hippocampus.

Action Potentials↗

PubMed2005

Changes of neuronal activity in areas CA1 and CA3 during anoxia and normoxic or hyperoxic reoxygenation in juvenile rat organotypic hippocampal slice cultures.

In neonates, asphyxia is usually followed by hyperoxic treatment. In order to study whether hyperoxic reoxygenation might cause additional impairment of neuronal function, we subjected organotypic hippocampal slice cultures of juvenile rats (7 DIV, P6-8) to 30 min anoxia followed by 60 min hyperoxic or normoxic reoxygenation (95% or 19% O2, respectively). Spontaneous and evoked field potentials as well as [Ca2+]o were recorded in the pyramidal layer of area CA1 or area CA3. In area CA1, 30 min of anoxia led to decline of evoked field potential amplitudes by on average 67% and to profound changes in field potential characteristics and Ca2+ homeostasis which were not related to outcome after reoxygenation. Hyperoxic reoxygenation resulted first in a fast recovery of the field potential amplitude to 82% of the control value and then, in 75% of slice cultures, in a large negative field potential shift accompanied by a prolonged decrease of [Ca2+]o and loss of excitability outlasting the experiment. Recovery of field potential amplitude under normoxic conditions stayed poor, with a first increase to 51% and a second decrease to 22%. In contrast, field potential amplitude in area CA3 recovered to 80% of the initial amplitude, irrespective of the reoxygenation mode. The selective loss of function during hyperoxic reoxygenation in area CA1 might be a first sign of neuronal injury that we observed 1 h after end of hyperoxic reoxygenation in a previous study. Whether the poor outcome after normoxic reoxygenation would favour long-term recovery remains to be determined.

Animals↗

PubMed2005

Neuronal Ca2+ sensor protein VILIP-1 affects cGMP signalling of guanylyl cyclase B by regulating clathrin-dependent receptor recycling in hippocampal neurons.

The family of neuronal Ca2+ sensor (NCS) proteins is known to influence a variety of physiological and pathological processes by affecting signalling of different receptors and ion channels. Recently, it has been shown that the NCS protein VILIP-1 influences the activity of the receptor guanylyl cyclase GC-B. In transfected cell lines, VILIP-1 performs a Ca2+-dependent membrane association, the reversible Ca2+-myristoyl switch of VILIP-1, which leads to an increase in natriuretic peptide-stimulated cGMP levels. In this study, we have investigated the effect of VILIP-1 on cGMP signalling in C6 cells and in primary hippocampal neurons, where VILIP-1 and GC-B are co-expressed in many but not all neurons and partially co-localize in the soma and in dendrites. Our data indicate that VILIP-1 modulates GC-B activity by influencing clathrin-dependent receptor recycling. These data support a general physiological role for VILIP-1 in membrane trafficking in the intact hippocampus, where the NCS protein may affect processes, such as neuronal differentiation and synaptic plasticity e.g. by influencing cGMP-signalling.

Animals↗

PubMed2005

Oxygen consumption and mitochondrial membrane potential indicate developmental adaptation in energy metabolism of rat cortical neurons.

Neuronal energy needs are mainly covered via mitochondrial oxidative phosphorylation. Even if the energy supply appears identical in immature and adult brain, nevertheless quantitative differences exist. The present study focuses on the adaptations in cellular energy metabolism caused by the neuronal maturation. As main parameters of oxidative phosphorylation, cellular oxygen consumption and mitochondrial membrane potential were measured in isolated rat cortical cells using a Clark-type oxygen electrode and microfluorometric techniques. In four age groups (E18-P2, P8-P12, P16-P20, > or = P28), unstimulated neurons showed a significant age-dependent increase in basal oxygen consumption (6.1 up to 10.2 nM/min/10(7) cells). The excitatory neurotransmitter glutamate induced a further, but age- and concentration-independent, elevation of oxygen consumption to a plateau > or = 14 nM/min/10(7) cells and a complete depolarization of mitochondrial membrane in neurons > or = P8. Stimulation using K+ (5-50 mM) effected a concentration- and age-dependent increase in oxygen consumption, but a similar nearby complete depolarization of mitochondrial membrane in all tested age groups. Furthermore, uncoupling mitochondrial membrane function followed by a complete depolarization of mitochondrial membrane showed a maximal oxygen consumption (14-15 nM/min/10(7) cells) only in neurons > or = P8. These data suggest that developing and adult cortical neurons cover their increased need of energy following stimulation by an efficiency improvement of mitochondrial oxidative phosphorylation. The age-independent limited capacity of mitochondrial oxidative phosphorylation, however, causes a reduction in cellular energy disposal in mature neurons and therefore may play a critical role in the increased sensitivity of adult neurons against excitotoxicity and ischaemia.

Animals↗

PubMed2005

Processes and components participating in the generation of intrinsic optical signal changes in vitro.

Imaging of intrinsic optical signals has become an important tool in the neurosciences. To better understand processes underlying changes in intrinsic optical signals, we studied electrical stimulation at varying strengths in hippocampal slices of adult Wistar rats. Following serial stimulation we observed an increase in light transmittance in all tested slices. During antidromic stimulation at minimum stimulation strength the increase in light transmittance was 75 +/- 8% (P < 0.05), and during orthodromic minimum stimulation 19.6 +/- 5.6% (P < 0.001) in the stratum pyramidale of the CA1-region. During orthodromic stimulation no significant difference between submaximum, maximum and supramaximum stimulation was found, indicating saturation. In contrast, submaximum antidromic stimulation yielded 56.2 +/- 12% (P < 0.05) of maximum stimulation strength, indicating recruitment. In a further set of experiments serial stimulation was carried out under glial blockade with fluoroacetate (FAC) or blockage of mitochondrial function. Amplitude and slope of the intrinsic optical signal significantly decreased in the presence of FAC (amplitude: 36 +/- 6%, P < 0.01; slope: 37 +/- 11% as compared with baseline conditions, P < 0.05). This suggests a glial participation in signal generation. Rotenone, an inhibitor of mitochondrial complex I, yielded decreased amplitudes of the intrinsic optical signal (27 +/- 7% after 40 min, P < 0.01). Our data indicate that the intrinsic optical signal change reflects type and strength of neuronal activation and point to glia and mitochondria as important participants in signal generation.

Action Potentials↗

PubMed2004

Recurrent seizures do not cause hippocampal damage.

Neuronal consequences of recurrent single epileptic seizures have been discussed controversially for some time. Various cross-sectional magnetic resonance imaging (MRI) studies have shown a positive correlation between the severity of epilepsy and the extent of hippocampal damage. However, the open question whether recurrent epileptic seizures induce hippocampal structural pathology can be assessed only in longitudinal studies. The few recent follow-up studies have revealed conflicting results. In the current MRI study we have employed volumetry and T2 relaxometry to quantify hippocampal structural changes of patients with chronic partial epilepsies over a period of 3 years. Our main findings demonstrate that these patients who experience continuing epileptic seizures do no show any development of new pathology or any relevant deterioration of pre-existing hippocampal structural lesions. This argues against the assumption that recurrent epileptic seizures cause or increase structural hippocampal damage.

Adult↗

PubMed2004

Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up-regulated after in vitro stimulation and in patients with rheumatoid arthritis.

Glucocorticoids mediate their therapeutic actions mostly by genomic effects via cytosolic receptors, but some effects are too rapid to be mediated by changes at the genomic level. The detailed mechanisms of these nongenomic actions are still unclear. Membrane-bound glucocorticoid receptors (mGCR) have been suggested to be involved, although their physiological existence in humans so far is hypothetical. For the first time we demonstrate the existence of mGCR on monocytes and B cells obtained from healthy blood donors using high-sensitivity immunofluorescent staining. Immunostimulation with lipopolysaccharide increases the percentage of mGCR-positive monocytes, which can be prevented by inhibiting the secretory pathway. Overexpression of the human glucocorticoid receptor alpha alone is not sufficient to enhance mGCR expression. These in vitro findings are consistent with our clinical observation that in patients with rheumatoid arthritis the frequency of mGCR positive monocytes is increased and positively correlated with disease activity. We conclude that mGCR are 1) indeed physiologically present in healthy blood donors, but remained unidentified by conventional techniques due to their small number per cell and 2) actively up-regulated and transported through the cell after immunostimulation. These receptors may reflect a feedback mechanism of the organism upon immunostimulation and/or play a role in pathogenesis.

Adolescent↗

PubMed2003

Quantitating protein synthesis, degradation, and endogenous antigen processing.

Using L929 cells, we quantitated the macroeconomics of protein synthesis and degradation and the microeconomics of producing MHC class I associated peptides from viral translation products. To maintain a content of 2.6 x 10(9) proteins, each cell's 6 x 10(6) ribosomes produce 4 x 10(6) proteins min(-1). Each of the cell's 8 x 10(5) proteasomes degrades 2.5 substrates min(-1), creating one MHC class I-peptide complex for each 500-3000 viral translation products degraded. The efficiency of complex formation is similar in dendritic cells and macrophages, which play a critical role in activating T cells in vivo. Proteasomes create antigenic peptides at different efficiencies from two distinct substrate pools: rapidly degraded newly synthesized proteins that clearly represent defective ribosomal products (DRiPs) and a less rapidly degraded pool in which DRiPs may also predominate.

Animals↗

PubMed2002

Erythropoietin improves synaptic transmission during and following ischemia in rat hippocampal slice cultures.

Erythropoietin (EPO) prevents neuronal damage following ischemic, metabolic, and excitotoxic stress. In this study evoked extracellular field potentials (FP) were used to investigate the effect of EPO on synaptic transmission in hippocampal slice cultures. EPO treated cultured slices (40 units/ml for 48 h) showed significantly increased FP during and following oxygen and glucose deprivation compared with untreated control slices. The addition of the Jak2 inhibitor AG490 (50 microM for 48 h) blocked the EPO effect. These data suggest that EPO improves synaptic transmission during and following ischemia in hippocampal slice cultures.

Animals↗

PubMed2002

Intrinsic optical imaging reveals regionally different manifestation of spreading depression in hippocampal and entorhinal structures in vitro.

The spatiotemporal features of spreading depression (SD) were analyzed in vitro by using combined hippocampal-entorhinal cortex slices. SDs were induced by microinjection of 1 M KCl in the stratum radiatum of the CA1 region of the hippocampus. Measurements of extracellular field potentials, extracellular space (ECS) volume changes and intrinsic optical signal changes were combined to study SD features in different regions of the slice. Each SD was associated with a pronounced shrinkage of the extracellular space (ECS) volume and a decrease in light transmittance. The beginning of the optical signal change occurred simultaneously with the electrographic onset as measured with extracellular microelectrodes but outlasted the dc shift for tens of seconds. The amplitude of the intrinsic optical signal change displayed marked regional variations with greatest changes of 12% in cortical regions. The signal amplitudes were considerably lower in hippocampal regions. The analysis of spread patterns revealed two types of waves: fully propagated waves spreading from CA1 all the way to the temporal neocortex and abortive waves that ceased earlier. The spread velocities displayed pronounced regional differences with highest velocities of 5.4 +/- 0.3 mm/min in the area CA3 of the hippocampal formation and lowest velocities of 2.7 +/- 0.1 mm/min in cortical regions.

Animals↗

PubMed2002

Nitric oxide modulates low-Mg2+-induced epileptiform activity in rat hippocampal-entorhinal cortex slices.

The production of nitric oxide (NO) during low-Mg2+-induced epileptiform activity in rat hippocampal-entorhinal cortex slices was investigated by real-time monitoring using 1,2-diaminoanthraquinone (DAQ). NO reacts with the aromatic amino groups of DAQ at neutral pH and in the presence of oxygen to form the fluorescence product 1H-anthra-[1,2d]-[1,2,3]triazole-6,11-dione (ATD). The DAQ-induced formation of ATD required NO and was insensitive to radical oxygen species. Removal of Mg2+ ions from the artificial cerebrospinal fluid (ACSF) induced a significant elevation in the ATD fluorescence signal. The application of L-arginine (2 mM), a substrate of nitric oxide synthase (NOS), caused a comparable increase in the ATD fluorescence signal. Furthermore, ATD signal increase induced either by low-Mg2+ ACSF or by L-arginine was sensitive to N-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor. The application of L-NAME (200 microM) caused a complete blockade of low-Mg2+-induced epileptiform activity. Under this condition, increasing NO concentration by addition of the NO donor S-nitroso-N-acetylpenicillamine (200 microM) reinduced the epileptiform activity. It has been concluded that onset and maintenance of low-Mg2+-induced spontaneous epileptiform activity are modulated by NO concentration. Further NO imaging studies may help to elucidate the role of NO in detail and may bring to light new means for epilepsy therapy.

Animals↗

PubMed2002

Spatial nitric oxide imaging using 1,2-diaminoanthraquinone to investigate the involvement of nitric oxide in long-term potentiation in rat brain slices.

Long-term potentiation (LTP), a model of activity-dependent synaptic plasticity, involves the persistent enhancement of excitatory neurotransmission. Several recent studies have suggested a critical role for nitric oxide (NO) production in hippocampal LTP. However, increase in NO production in living tissue has not yet been directly demonstrated. We used 1,2-diaminoanthraquinone (DAQ) to demonstrate NO production in rat brain slices in relation to induction of LTP. DAQ was found to be without neurotoxic effects and it neither influenced normal evoked field potential amplitudes nor did it affect induction of LTP in comparison to controls. We found that DAQ-induced fluorescence is elevated within a limited area of about 40,000 microm(2) during LTP induction in the hippocampal area CA1. Furthermore, we could demonstrate that application of the NO-synthetase inhibitor l-NAME inhibits the induction of LTP in area CA1 and causes a strong reduction of DAQ induced fluorescence. Our results are consistent with the hypothesis that NO can serve as a retrograde messenger during induction of LTP in the hippocampus.

Animals↗

PubMed2002

Free radical-mediated cell damage after experimental status epilepticus in hippocampal slice cultures.

Generation of free radicals may have a key role in the nerve cell damage induced by prolonged or frequently recurring convulsions (status epilepticus). Mitochondrial function may also be altered due to production of free radicals during seizures. We therefore studied changes in field potentials (fp) together with measurements of extracellular, intracellular, and intramitochondrial calcium concentration ([Ca(2+)]e, [Ca(2+)]i, and [Ca(2+)]m, respectively), mitochondrial membrane potential (deltapsi), NAD(P)H auto-fluorescence, and dihydroethidium (HEt) fluorescence in hippocampal slice cultures by means of simultaneous electrophysiological and microfluorimetric measurements. As reported previously, each seizure-like event (SLE) resulted in mitochondrial depolarization associated with a delayed rise in oxidation of HEt to ethidum, presumably indicating ROS production. We show here that repeated SLEs led to a decline in intracellular and intramitochondrial Ca(2+) signals despite unaltered Ca(2+) influx. Also, mitochondrial depolarization and the NAD(P)H signal became smaller during recurring SLEs. By contrast, the ethidium fluorescence rises remained constant or even increased from SLE to SLE. After about 15 SLEs, activity changed to continuous afterdischarges with steady depolarization of mitochondrial membranes. Staining with a cell death marker, propidium iodide, indicated widespread cell damage after 2 h of recurring SLEs. The free radical scavenger, alpha-tocopherol, protected the slice cultures against this damage and also reduced the ongoing impairment of NAD(P)H production. These findings suggest involvement of reactive oxygen species (ROS) of mitochondrial origin in the epileptic cell damage and that free radical scavenging may prevent status epilepticus-induced cell loss.

Animals↗