Although absence epilepsy has a genetic origin, evidence from an animal model (Wistar Albino Glax... more Although absence epilepsy has a genetic origin, evidence from an animal model (Wistar Albino Glaxo/Rijswijk; WAG/Rij) suggests that seizures are sensitive to environmental manipulations. Here, we show that manipulations of the early rearing environment (neonatal handling, maternal deprivation) of WAG/Rij rats leads to a pronounced decrease in seizure activity later in life. Recent observations link seizure activity in WAG/Rij rats to the hyperpolarization-activated cation current (Ih) in the somatosensory cortex, the site of seizure generation. Therefore, we investigated whether the alterations in seizure activity between rats reared differently might be correlated with changes in Ih and its channel subunits hyperpolarization-activated cation channel HCN1, 2 and 4. Whole-cell recordings from layer 5 pyramidal neurons, in situ hybridization and Western blot of the somatosensory cortex revealed an increase in Ih and HCN1 in neonatal handled and maternal deprived, compared to control rats. The increase was specific to HCN1 protein expression and did not involve HCN2/4 protein expression, or mRNA expression of any of the subunits (HCN1, 2, 4). Our findings provide the first evidence that relatively mild changes in the neonatal environment have a long-term impact of absence seizures, Ih and HCN1, and suggest that an increase of Ih and HCN1 is associated with absence seizure reduction. Our findings shed new light on the role of Ih and HCN in brain functioning and development and demonstrate that genetically determined absence seizures are quite sensitive for early interventions.
The Journal of Trauma: Injury, Infection, and Critical Care, 2007
Prolonged ischemia followed by reperfusion (I/R) of skeletal muscle results in significant tissue... more Prolonged ischemia followed by reperfusion (I/R) of skeletal muscle results in significant tissue injury. Ischemic preconditioning (IPC), achieved by brief periods of ischemia before sustained ischemia, has been shown to ameliorate I/R injury in a variety of tissues. We demonstrate that tourniquet hind limb ischemia-induced injury of the muscle benefits from IPC, whereas the peripheral nerve suffers from prolonged ischemia time and mechanical deterioration on IPC. In anesthetized rats, hind limb ischemia was induced by tourniquet for 3 hours followed by 24 hours of reperfusion. In an additional series of experiments, IPC (three cycles of 10 minutes I/10 minutes R) preceded hind limb ischemia. Sham-operated animals without ischemia served as controls. Skeletal muscle tissue injury was assessed with respect to microcirculation, inflammatory cell response, and cell integrity using intravital fluorescence microscopy, Western blot protein analysis, and tissue histochemistry. Analysis of tactile and thermal allodynia served as indicators for postischemic pain. In addition, motor nerve conduction velocity and transmission electron microscopy allowed assessing postischemic nerve lesion. Tourniquet of the hind limb caused marked perfusion failure, enhanced leukocyte-endothelial cell interaction, and apoptotic cell death. IPC was able to improve microvascular perfusion and to reduce inflammatory cell response. Of interest, apoptotic cell death, assessed by cell nuclear morphology in vivo as well as Western blot and immunohistochemical analysis of caspase-3 cleavage, can be substantially reduced by IPC in tourniquet ischemia of the hind limb. Application of the tourniquet abolished nerve conduction in all animals. Non-IPC-treated animals still showed tactile allodynia, whereas IPC further caused loss of pain sensation and motor function of the postischemic hind limb. High susceptibility of the peripheral nerve to compression-induced ischemic injury disproves IPC in its clinical application for surgical procedures requiring prolonged tourniquet ischemia.
Neuropsychiatric developmental disorders, such as autism spectrum disorders (ASDs) and schizophre... more Neuropsychiatric developmental disorders, such as autism spectrum disorders (ASDs) and schizophrenia, are typically characterized by alterations in social behavior and have been linked to aberrant dendritic spine and synapse development. Here we show, using genetically engineered mice, that the Cdc42 GTPase-activating multiadaptor protein, NOMA-GAP, regulates autism-like social behavior in the mouse, as well as dendritic spine and synapse development. Surprisingly, we were unable to restore spine morphology or autism-associated social behavior in NOMA-GAP-deficient animals by Cre-mediated deletion of Cdc42 alone. Spine morphology can be restored in vivo by re-expression of wild-type NOMA-GAP or a mutant of NOMA-GAP that lacks the RhoGAP domain, suggesting that other signaling functions are involved. Indeed, we show that NOMA-GAP directly interacts with several MAGUK (membrane-associated guanylate kinase) proteins, and that this modulates NOMA-GAP activity toward Cdc42. Moreover, we ...
Cytokines are key players in the interactions of the immune and nervous systems. Recently, we sho... more Cytokines are key players in the interactions of the immune and nervous systems. Recently, we showed that such interplay is mediated by type I interferons (IFNs), which elevate the excitability of neocortical pyramidal neurons. A line of indirect evidence suggested that modulation of multiple ion channels underlies the effect. However, which currents are principally involved and how the IFN signaling cascade is linked to the respective ion channels remains elusive. We tested several single and combined ionic current modulations using an in silico model of a neocortical layer 5 neuron. Subsequently we investigated resulting predictions by whole-cell patch-clamp recordings in layer 5 neurons of ex vivo neocortical rat brain slices pharmacologically reproducing or prohibiting neuronal IFN effects. The amount and type of modulation necessary to replicate IFN effects in silico suggested protein kinase C (PKC) activation as link between the type I IFN signaling and ion channel modulations...
The transmembrane protein plasticity-related genes 3 and 5 (PRG3 and PRG5) increase filopodial fo... more The transmembrane protein plasticity-related genes 3 and 5 (PRG3 and PRG5) increase filopodial formation in various cell lines, independently of Cdc42. However, information on the effects of PRG5 during neuronal development is sparse. Here, we present several lines of evidence for the involvement of PRG5 in the genesis and stabilization of dendritic spines. First, PRG5 was strongly expressed during mouse brain development from embryonic day 14 (E14), peaked around the time of birth, and remained stable at least until early adult stages (i.e. P30). Second, on a subcellular level, PRG5 expression shifted from an equal distribution along all neurites toward accumulation only along dendrites during hippocampal development in vitro. Third, overexpression of PRG5 in immature hippocampal neurons induced formation of spine-like structures ahead of time. Proper amino acid sequences in the extracellular domains (D1 to D3) of PRG5 were a prerequisite for trafficking and induction of spine-like structures, as shown by mutation analysis. Fourth, at stages when spines are present, knockdown of PRG5 reduced the number but not the length of protrusions. This was accompanied by a decrease in the number of excitatory synapses and, consequently, by a reduction of miniature excitatory postsynaptic current frequencies, although miniature excitatory postsynaptic current amplitudes remained similar. In turn, overexpressing PRG5 in mature neurons not only increased Homer-positive spine numbers but also augmented spine head diameters. Mechanistically, PRG5 interacts with phosphorylated phosphatidylinositols, phospholipids involved in dendritic spine formation by different lipid-protein assays. Taken together, our data propose that PRG5 promotes spine formation.
Biochemical and Biophysical Research Communications, 1996
The effect of cicutoxin, the poisonous principle of the genusCicuta, on K+currents of activated T... more The effect of cicutoxin, the poisonous principle of the genusCicuta, on K+currents of activated T lymphocytes was investigated using the patch clamp technique. Cicutoxin produced a dose-dependent [5 × 10− 6–7 × 10−5mol/l] and completely reversible block of K+currents with an EC50of 1.8 × 10−5mol/l. A maximum block of 71% was achieved with cicutoxin at a concentration of 7 ×
The immunomodulatory cytokine interferon-beta (IFN-beta) is used in the treatment of autoimmune d... more The immunomodulatory cytokine interferon-beta (IFN-beta) is used in the treatment of autoimmune diseases such as multiple sclerosis. However, the effect of IFN-beta on neuronal functions is currently unknown. Intracellular recordings were conducted on somatosensory neurons of neocortical layers 2/3 and 5 exposed to IFN-beta. The excitability of neurons was increased by IFN-beta (10-10,000 U/ml) in two kinetically distinct, putatively independent manners. First IFN-beta reversibly influenced the subthreshold membrane response by raising the membrane resistance R(M) 2.5-fold and the membrane time constant tau 1.7-fold dose-dependently. The effect required permanent exposure to IFN-beta and was reduced in magnitude if the extracellular K+ was lowered. However, the membrane response to IFN-beta in the subthreshold range was prevented by ZD7288 (a specific blocker of I(h)) but not by Ni2+, carbachol, or bicuculline, pointing to a dependence on an intact I(h). Second, IFN-beta enhanced the rate of action potential firing. This effect was observed to develop for >1 h when the cell was exposed to IFN-beta for 5 min or >5 min and showed no reversibility (< or =210 min). Current-discharge (F-I) curves revealed a shift (prevented by bicuculline) as well as an increase in slope (prevented by carbachol and Ni2+). Layer specificity was not observed with any of the described effects. In conclusion, IFN-beta influences the neuronal excitability in neocortical pyramidal neurons in vitro, especially under conditions of slightly increased extracellular K+. Our blocker experiments indicate that changes in various ionic conductances with different voltage dependencies cause different IFN-beta influences on sub- and suprathreshold behavior, suggesting a more general intracellular process induced by IFN-beta.
Pfl�gers Archiv European Journal of Physiology, 2001
Perforated whole-cell patch-clamp recordings obtained with nystatin are frequently used to preser... more Perforated whole-cell patch-clamp recordings obtained with nystatin are frequently used to preserve intracellular integrity. However, the perforated-patch configuration may sometimes undergo a spontaneous change into the conventional whole-cell configuration, especially when lymphocytes are investigated. The electrophysiological criteria-- previously described--for establishing the existence of the perforated whole-cell configuration have been shown to be insufficient. Thus, the dye eosin, applied to the pipette solution, was tested as a tool for discriminating between the perforated and the conventional whole-cell configurations on rat T-lymphocytes. The dye never entered the cell from the pipette during the entire measurement in the perforated whole-cell configuration. In contrast, all cells in the conventional whole-cell configuration became red immediately after membrane rupture. Eosin barely changed the currents studied. The results suggest that eosin is a dye of choice for verifying a true perforated-patch configuration.
Vast electrophysiological activity near resting potential, including rhythmic oscillatory activit... more Vast electrophysiological activity near resting potential, including rhythmic oscillatory activity, is a hallmark of many brain regions and a motor of the developing CNS. This activity is mediated and influenced by diverse receptor-operated and voltage-gated ion channels. In turn, these channels are modulated during the course of development by altering their density, distribution and properties. The hyperpolarization-activated and cyclic nucleotide-gated cation current, Ih, impacts on the resting membrane potential and is involved in the generation and modulation of neuronal oscillatory activity. Therefore, it is conceivable that Ih is well suited to govern the specific processes involved in activity-dependent neuronal development. Here, we review the evidence that maturation of Ih accounts, at least in part, for the control of membrane properties during neuronal development of various parts of the brain. The temporal and regional variations in Ih development might underlie the normal maturation of neuronal circuits and, consequently, the perturbations of this might account for some of the neuropathology of the brain. This review summarizes the evidence for the stage and localization dependence of Ih in CNS development with a focus on arborized cells with high dendritic Ih. Further, it outlines hypotheses on the contribution of Ih to neuronal and network maturation.
Although absence epilepsy has a genetic origin, evidence from an animal model (Wistar Albino Glax... more Although absence epilepsy has a genetic origin, evidence from an animal model (Wistar Albino Glaxo/Rijswijk; WAG/Rij) suggests that seizures are sensitive to environmental manipulations. Here, we show that manipulations of the early rearing environment (neonatal handling, maternal deprivation) of WAG/Rij rats leads to a pronounced decrease in seizure activity later in life. Recent observations link seizure activity in WAG/Rij rats to the hyperpolarization-activated cation current (Ih) in the somatosensory cortex, the site of seizure generation. Therefore, we investigated whether the alterations in seizure activity between rats reared differently might be correlated with changes in Ih and its channel subunits hyperpolarization-activated cation channel HCN1, 2 and 4. Whole-cell recordings from layer 5 pyramidal neurons, in situ hybridization and Western blot of the somatosensory cortex revealed an increase in Ih and HCN1 in neonatal handled and maternal deprived, compared to control rats. The increase was specific to HCN1 protein expression and did not involve HCN2/4 protein expression, or mRNA expression of any of the subunits (HCN1, 2, 4). Our findings provide the first evidence that relatively mild changes in the neonatal environment have a long-term impact of absence seizures, Ih and HCN1, and suggest that an increase of Ih and HCN1 is associated with absence seizure reduction. Our findings shed new light on the role of Ih and HCN in brain functioning and development and demonstrate that genetically determined absence seizures are quite sensitive for early interventions.
The Journal of Trauma: Injury, Infection, and Critical Care, 2007
Prolonged ischemia followed by reperfusion (I/R) of skeletal muscle results in significant tissue... more Prolonged ischemia followed by reperfusion (I/R) of skeletal muscle results in significant tissue injury. Ischemic preconditioning (IPC), achieved by brief periods of ischemia before sustained ischemia, has been shown to ameliorate I/R injury in a variety of tissues. We demonstrate that tourniquet hind limb ischemia-induced injury of the muscle benefits from IPC, whereas the peripheral nerve suffers from prolonged ischemia time and mechanical deterioration on IPC. In anesthetized rats, hind limb ischemia was induced by tourniquet for 3 hours followed by 24 hours of reperfusion. In an additional series of experiments, IPC (three cycles of 10 minutes I/10 minutes R) preceded hind limb ischemia. Sham-operated animals without ischemia served as controls. Skeletal muscle tissue injury was assessed with respect to microcirculation, inflammatory cell response, and cell integrity using intravital fluorescence microscopy, Western blot protein analysis, and tissue histochemistry. Analysis of tactile and thermal allodynia served as indicators for postischemic pain. In addition, motor nerve conduction velocity and transmission electron microscopy allowed assessing postischemic nerve lesion. Tourniquet of the hind limb caused marked perfusion failure, enhanced leukocyte-endothelial cell interaction, and apoptotic cell death. IPC was able to improve microvascular perfusion and to reduce inflammatory cell response. Of interest, apoptotic cell death, assessed by cell nuclear morphology in vivo as well as Western blot and immunohistochemical analysis of caspase-3 cleavage, can be substantially reduced by IPC in tourniquet ischemia of the hind limb. Application of the tourniquet abolished nerve conduction in all animals. Non-IPC-treated animals still showed tactile allodynia, whereas IPC further caused loss of pain sensation and motor function of the postischemic hind limb. High susceptibility of the peripheral nerve to compression-induced ischemic injury disproves IPC in its clinical application for surgical procedures requiring prolonged tourniquet ischemia.
Neuropsychiatric developmental disorders, such as autism spectrum disorders (ASDs) and schizophre... more Neuropsychiatric developmental disorders, such as autism spectrum disorders (ASDs) and schizophrenia, are typically characterized by alterations in social behavior and have been linked to aberrant dendritic spine and synapse development. Here we show, using genetically engineered mice, that the Cdc42 GTPase-activating multiadaptor protein, NOMA-GAP, regulates autism-like social behavior in the mouse, as well as dendritic spine and synapse development. Surprisingly, we were unable to restore spine morphology or autism-associated social behavior in NOMA-GAP-deficient animals by Cre-mediated deletion of Cdc42 alone. Spine morphology can be restored in vivo by re-expression of wild-type NOMA-GAP or a mutant of NOMA-GAP that lacks the RhoGAP domain, suggesting that other signaling functions are involved. Indeed, we show that NOMA-GAP directly interacts with several MAGUK (membrane-associated guanylate kinase) proteins, and that this modulates NOMA-GAP activity toward Cdc42. Moreover, we ...
Cytokines are key players in the interactions of the immune and nervous systems. Recently, we sho... more Cytokines are key players in the interactions of the immune and nervous systems. Recently, we showed that such interplay is mediated by type I interferons (IFNs), which elevate the excitability of neocortical pyramidal neurons. A line of indirect evidence suggested that modulation of multiple ion channels underlies the effect. However, which currents are principally involved and how the IFN signaling cascade is linked to the respective ion channels remains elusive. We tested several single and combined ionic current modulations using an in silico model of a neocortical layer 5 neuron. Subsequently we investigated resulting predictions by whole-cell patch-clamp recordings in layer 5 neurons of ex vivo neocortical rat brain slices pharmacologically reproducing or prohibiting neuronal IFN effects. The amount and type of modulation necessary to replicate IFN effects in silico suggested protein kinase C (PKC) activation as link between the type I IFN signaling and ion channel modulations...
The transmembrane protein plasticity-related genes 3 and 5 (PRG3 and PRG5) increase filopodial fo... more The transmembrane protein plasticity-related genes 3 and 5 (PRG3 and PRG5) increase filopodial formation in various cell lines, independently of Cdc42. However, information on the effects of PRG5 during neuronal development is sparse. Here, we present several lines of evidence for the involvement of PRG5 in the genesis and stabilization of dendritic spines. First, PRG5 was strongly expressed during mouse brain development from embryonic day 14 (E14), peaked around the time of birth, and remained stable at least until early adult stages (i.e. P30). Second, on a subcellular level, PRG5 expression shifted from an equal distribution along all neurites toward accumulation only along dendrites during hippocampal development in vitro. Third, overexpression of PRG5 in immature hippocampal neurons induced formation of spine-like structures ahead of time. Proper amino acid sequences in the extracellular domains (D1 to D3) of PRG5 were a prerequisite for trafficking and induction of spine-like structures, as shown by mutation analysis. Fourth, at stages when spines are present, knockdown of PRG5 reduced the number but not the length of protrusions. This was accompanied by a decrease in the number of excitatory synapses and, consequently, by a reduction of miniature excitatory postsynaptic current frequencies, although miniature excitatory postsynaptic current amplitudes remained similar. In turn, overexpressing PRG5 in mature neurons not only increased Homer-positive spine numbers but also augmented spine head diameters. Mechanistically, PRG5 interacts with phosphorylated phosphatidylinositols, phospholipids involved in dendritic spine formation by different lipid-protein assays. Taken together, our data propose that PRG5 promotes spine formation.
Biochemical and Biophysical Research Communications, 1996
The effect of cicutoxin, the poisonous principle of the genusCicuta, on K+currents of activated T... more The effect of cicutoxin, the poisonous principle of the genusCicuta, on K+currents of activated T lymphocytes was investigated using the patch clamp technique. Cicutoxin produced a dose-dependent [5 × 10− 6–7 × 10−5mol/l] and completely reversible block of K+currents with an EC50of 1.8 × 10−5mol/l. A maximum block of 71% was achieved with cicutoxin at a concentration of 7 ×
The immunomodulatory cytokine interferon-beta (IFN-beta) is used in the treatment of autoimmune d... more The immunomodulatory cytokine interferon-beta (IFN-beta) is used in the treatment of autoimmune diseases such as multiple sclerosis. However, the effect of IFN-beta on neuronal functions is currently unknown. Intracellular recordings were conducted on somatosensory neurons of neocortical layers 2/3 and 5 exposed to IFN-beta. The excitability of neurons was increased by IFN-beta (10-10,000 U/ml) in two kinetically distinct, putatively independent manners. First IFN-beta reversibly influenced the subthreshold membrane response by raising the membrane resistance R(M) 2.5-fold and the membrane time constant tau 1.7-fold dose-dependently. The effect required permanent exposure to IFN-beta and was reduced in magnitude if the extracellular K+ was lowered. However, the membrane response to IFN-beta in the subthreshold range was prevented by ZD7288 (a specific blocker of I(h)) but not by Ni2+, carbachol, or bicuculline, pointing to a dependence on an intact I(h). Second, IFN-beta enhanced the rate of action potential firing. This effect was observed to develop for >1 h when the cell was exposed to IFN-beta for 5 min or >5 min and showed no reversibility (< or =210 min). Current-discharge (F-I) curves revealed a shift (prevented by bicuculline) as well as an increase in slope (prevented by carbachol and Ni2+). Layer specificity was not observed with any of the described effects. In conclusion, IFN-beta influences the neuronal excitability in neocortical pyramidal neurons in vitro, especially under conditions of slightly increased extracellular K+. Our blocker experiments indicate that changes in various ionic conductances with different voltage dependencies cause different IFN-beta influences on sub- and suprathreshold behavior, suggesting a more general intracellular process induced by IFN-beta.
Pfl�gers Archiv European Journal of Physiology, 2001
Perforated whole-cell patch-clamp recordings obtained with nystatin are frequently used to preser... more Perforated whole-cell patch-clamp recordings obtained with nystatin are frequently used to preserve intracellular integrity. However, the perforated-patch configuration may sometimes undergo a spontaneous change into the conventional whole-cell configuration, especially when lymphocytes are investigated. The electrophysiological criteria-- previously described--for establishing the existence of the perforated whole-cell configuration have been shown to be insufficient. Thus, the dye eosin, applied to the pipette solution, was tested as a tool for discriminating between the perforated and the conventional whole-cell configurations on rat T-lymphocytes. The dye never entered the cell from the pipette during the entire measurement in the perforated whole-cell configuration. In contrast, all cells in the conventional whole-cell configuration became red immediately after membrane rupture. Eosin barely changed the currents studied. The results suggest that eosin is a dye of choice for verifying a true perforated-patch configuration.
Vast electrophysiological activity near resting potential, including rhythmic oscillatory activit... more Vast electrophysiological activity near resting potential, including rhythmic oscillatory activity, is a hallmark of many brain regions and a motor of the developing CNS. This activity is mediated and influenced by diverse receptor-operated and voltage-gated ion channels. In turn, these channels are modulated during the course of development by altering their density, distribution and properties. The hyperpolarization-activated and cyclic nucleotide-gated cation current, Ih, impacts on the resting membrane potential and is involved in the generation and modulation of neuronal oscillatory activity. Therefore, it is conceivable that Ih is well suited to govern the specific processes involved in activity-dependent neuronal development. Here, we review the evidence that maturation of Ih accounts, at least in part, for the control of membrane properties during neuronal development of various parts of the brain. The temporal and regional variations in Ih development might underlie the normal maturation of neuronal circuits and, consequently, the perturbations of this might account for some of the neuropathology of the brain. This review summarizes the evidence for the stage and localization dependence of Ih in CNS development with a focus on arborized cells with high dendritic Ih. Further, it outlines hypotheses on the contribution of Ih to neuronal and network maturation.
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Papers by Ulf Strauss