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.
Studying axonal outgrowth and regeneration of the corticospinal tract in organotypic slice cultur... more Studying axonal outgrowth and regeneration of the corticospinal tract in organotypic slice cultures (
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 distribution of ion channels in neurons regulates neuronal activity and proper formation of n... more The distribution of ion channels in neurons regulates neuronal activity and proper formation of neuronal networks during neuronal development. One of the channels is the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel constituting the molecular substrate of hyperpolarization-activated current (I h ). Our previous study implied a role for the fastest activating subunit HCN1 in the generation of I h in rat neonatal cortical plate neurons. To better understand the impact of HCN1 in early neocortical development, we here performed biochemical analysis and whole-cell recordings in neonatal cortical plate and juvenile layer 5 somatosensory neurons of HCN1 2/2 and control HCN1 1/1 mice. Western Blot analysis revealed that HCN1 protein expression in neonatal cortical plate tissue of HCN 1/1 mice amounted to only 3% of the HCN1 in young adult cortex and suggested that in HCN1 2/2 mice other isoforms (particularly HCN4) might be compensa-tory up-regulated. At the first day after birth, functional ablation of the HCN1 subunit did not affect the proportion of I h expressing pyramidal cortical plate neurons. Although the contribution of individual subunit proteins remains open, the lack of HCN1 markedly slowed the current activation and deactivation in individual I h expressing neurons. However, it did not impair maximal amplitude/density, voltage dependence of activation, and cAMP sensitivity. In conclusion, our data imply that, although expression is relatively low, HCN1 contributes substantially to I h properties in individual cortical plate neurons. These properties are significantly changed in HCN1 2/2 , either due to the lack of HCN1 itself or due to compensatory mechanisms.
Background/Aims: Hyperpolarization activated cyclic nucleotide gated 1 (HCN1) channels determine ... more Background/Aims: Hyperpolarization activated cyclic nucleotide gated 1 (HCN1) channels determine neuronal excitability in several brain regions. In contrast to HCN2 and HCN4, HCN1 is less sensitive to cAMP and the number of other known modulators is limited. One of those, the protein kinase C (PKC), showed opposing effects on mouse HCN1 channels expressed in Xenopus oocytes. Methods: In order to study PKC effects on HCN1 mediated currents in a mammalian environment we expressed rat HCN1 or human HCN1 in human embryonic kidney (HEK293) cells and rat HCN1 in murine neuroblastoma (N1E-115) cells. We recorded the resulting I h before and during the application of the membrane permeable nonmetabolizable PKC-activator 4βPMA in cell-attached mode of the patch-clamp technique, leaving the intracellular environment intact. Results: 4βPMA reduced maximal HCN1 mediated currents to about 60-70 % and slowed its activation, but left its voltage sensitivity unchanged. The effect was neither due to species-related differences nor restricted to HEK293 cells, because it was comparable for human and rat HCN1 in HEK293 and for rat HCN1 in N1E-115 cells. However, pre-treatment with the PKC blocker GF109203X abolished 4βPMA induced I h changes. Disrupting the intracellular environment by recording in whole-cell mode drastically reduced the 4βPMA effect. Conclusion: PKC activation reduces and slows I h in non-neuronal and neuronal mammalian cells transfected with rat or human HCN1 if the intracellular content remains intact. 535 Reetz/Strauss: PKC Mediated HCN1 Inhibition Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry P = 0.3; /F under 1 µM 4βPMA or V 1/2ctrl = -98.0 ± 3.0 mV vs. V 1/24βPMA = -101.9 ± 1.7 mV n = 8, paired t-test: P = 0.07 under 0.2 µM 4βPMA). These data suggest that activation of PKC results in a suppression of maximal rHCN1 mediated current accompanied by a slowing of its activation.
Quantitative trait loci (QTL) analysis has been carried out to identify genes controlling aluminu... more Quantitative trait loci (QTL) analysis has been carried out to identify genes controlling aluminum tolerance in rice. One hundred and six doubled haploid plants derived from a cross between a japonica variety, Azucena, and an indica variety, IR64, were used for QTL mapping and 175 RFLP and isozyme markers were employed to identify QTLs. QTL analysis revealed the presence of two QTLs, QTL 1 and QTL 2, which located in the middle of chromosome 6 and at the end of chromosome 8, respectively. The two QTLs explained 21.6% of the total phenotypic variation in the population based on a multiple regression model.
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 ×
At present, the most efficient therapeutical treatment of multiple sclerosis (MS) is achieved by ... more At present, the most efficient therapeutical treatment of multiple sclerosis (MS) is achieved by IFN-ß. However, its in vivo effects remain incompletely understood. If applied parenterally, the hydrophobic IFN-ß acts primarilyon blood cells with probable selectivity tor functionally different lymphocyte subpopulations, monocytes and granulocytes. We have investi!ated the expression of the activation marker interleukin-2 receptor-a (CD25) on CD3+ T cells, CD19 B cells, foetal-type 'Y8+CD3+ T cells and foetal-type CD5+CD19+ B cells of the peripheral blood. In addition, the oxidative burst activity and apoptosis have been determined in mononuclear and polymorphonuclear blood cells, respectively. The study accompanied aphase rn trial with IFN-ßlb (BETAFERON~, Schering). Two groups ofMS patients with relapsing-remitting course of the disease have been investigated at 8 time points (days 0, 5, 15,31,60,90, 180 and 270 after starting therapy): (1) verum group (n = 8) with application of8 Mill. units IFN-ßlb every other day, and (2) placebo group (n = 4) with application ofplacebo fOT 3 months and therapy as in (1) from day 90 onward. The main results were: (1) Activated T cells decreased until day 180 in the verum group and return thereafter to pre-treatment values, whereas in the placebo group the values remained relatively stahle over the whole observation period. (2) Activated B cells increased between days 90 and 270 in both groups, i.e. after verum application in both groups. (3) Foetal-type B cells were more activated than total B and T cells with increase over time in both groups.
Neuronal progenitor cells delivering neurotrophic factors are a promising therapeutic tool for tr... more Neuronal progenitor cells delivering neurotrophic factors are a promising therapeutic tool for treatment of neurodegenerative diseases. Although several promising results have come from studies in different animal models, detailed knowledge of the action of neurotrophic factors in the CNS is still lacking. A clonally derived, immortalized rat striatal cell line (ST14A) expressing ciliary neurotrophic factor (CNTF) offers a stable and controlled background with which to analyze CNTF actions on the transcriptional level in CNS progenitor cells. To identify early transcriptional changes induced by CNTF expression, we transfected the CNTF gene into ST14A cells, which differentiate at the nonpermissive temperature of 39°C via suppression of the immortalizing SV40 large T antigen. This shows a CNTF-dependent hypoxic/ ischemic stress response during the earliest stage of differentiation, with expression of specific transcripts and evidence of translational repression leading to decreased protein synthesis in the transfected cells. This process is mediated by the Ras/MAP kinase pathway and is accompanied by impaired proliferation and metabolism as well as signs of neuronal differentiation. The stress-like response in the early stage of differentiation improves the ability of the transfected cells to respond to and cope with a stressful environment in vivo. The present data indicate higher viability, longer life, and greater differentiation capacity of CNTF-ST14A cells if they are used for transplantation. We conclude that the stress-like response during the early stage of differentiation improves the ability of the CNTF-ST14A cells to respond and adapt to a stressful environment, which renders them useful candidate cells for in vivo trials of treatment for neurodegenerative diseases in animal models, e.g., of Huntington's disease.
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.
Deep brain stimulation (DBS) has become a treatment for a growing number of neurological and psyc... more Deep brain stimulation (DBS) has become a treatment for a growing number of neurological and psychiatric disorders, especially for therapy-refractory Parkinson's disease (PD). However, not all of the symptoms of PD are sufficiently improved in all patients, and side effects may occur. Further progress depends on a deeper insight into the mechanisms of action of DBS in the context of disturbed brain circuits. For this, optimized animal models have to be developed. We review not only charge transfer mechanisms at the electrode/tissue interface and strategies to increase the stimulation's energy-efficiency but also the electrochemical, electrophysiological, biochemical and functional effects of DBS. We introduce a hemi-Parkinsonian rat model for long-term experiments with chronically instrumented rats carrying a backpack stimulator and implanted platinum/iridium electrodes. This model is suitable for (1) elucidating the electrochemical processes at the electrode/tissue interface, (2) analyzing the molecular, cellular and behavioral stimulation effects, (3) testing new target regions for DBS, (4) screening for potential neuroprotective DBS effects, and (5) improving the efficacy and safety of the method. An outlook is given on further developments of experimental DBS, including the use of transgenic animals and the testing of closed-loop systems for the direct on-demand application of electric stimulation.
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.
In rat models of Parkinson's disease, injections of 6-hydroxydopamine (6-OHDA) into different are... more In rat models of Parkinson's disease, injections of 6-hydroxydopamine (6-OHDA) into different areas of the basal ganglia result in dopaminergic neurodegeneration in the substantia nigra. The extent and time course of the dopaminergic lesions varies between the models. While the effects on neurons have been extensively studied, little is known about the effects on astrocytes. We compared astrocytic activation (i.e. increase in number and staining intensity of glial fibrillary acidic protein immunoreactive cells) at the injection site and in downstream structures of the motor loop, i.e. the globus pallidus (GP) and the subthalamic nucleus (STN) following 6-OHDA lesion of the medial forebrain bundle (MFB) or the striatum. Lesions in both regions resulted in astrocytic activation at the lesion site, but their remote effects varied. MFB injections caused astrocytic activation in the ipsi-and contralateral striatum, whereas striatal injections resulted in astrocytic activation in the GP and STN. Since 6-OHDA injections into the MFB and the striatum result in complete and partial SNc lesions, respectively, we hypothesize that communication links exist between astrocytes, or between neurons and astrocytes, along neuronal pathways that transmit activating signals in response to neuronal damage-but only if the neuronal pathways are at least partially intact. ß
Apart from a central function in the extrapyramidal motor system, dopamine has been suggested to ... more Apart from a central function in the extrapyramidal motor system, dopamine has been suggested to play a role in neuroimmune interactions. Particularly in diseases of the central nervous system, such as multiple sclerosis, alterations in dopamine homeostasis might have immunological consequences. We investigated potential effects of dopamine stabilized by ascorbic acid on specifically activated encephalitogenic T cells at the peak of activation. Those cells exhibited an upregulation of voltage-sensitive K + channels which play a role in many neurotransmitter responses of lymphocytes and fulfilled a prerequisite to respond to dopamine, i.e. stable expression of mRNA for dopamine receptors DRD1, DRD2 and DRD3. However, whole-cell and perforated whole-cell recordings revealed no change in voltage-sensitive K + currents. Moreover, T cell proliferation was not changed in the presence of dopamine. Previously reported dopamine effects on T cells may be explained by a comparatively lower activation of the cells under investigation, suggesting an activation dependence of dopamine effects that may not be mediated by K + channels. Alternatively, the occurrence of dopamine degradation products under unprotected conditions may account for the changes reported. Nevertheless, care should be taken when using the dopamine-protecting anti-oxidant ascorbic acid, since we found that it markedly inhibited both K + currents and lymphocyte proliferation at higher concentrations.
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.
Studying axonal outgrowth and regeneration of the corticospinal tract in organotypic slice cultur... more Studying axonal outgrowth and regeneration of the corticospinal tract in organotypic slice cultures (
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 distribution of ion channels in neurons regulates neuronal activity and proper formation of n... more The distribution of ion channels in neurons regulates neuronal activity and proper formation of neuronal networks during neuronal development. One of the channels is the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel constituting the molecular substrate of hyperpolarization-activated current (I h ). Our previous study implied a role for the fastest activating subunit HCN1 in the generation of I h in rat neonatal cortical plate neurons. To better understand the impact of HCN1 in early neocortical development, we here performed biochemical analysis and whole-cell recordings in neonatal cortical plate and juvenile layer 5 somatosensory neurons of HCN1 2/2 and control HCN1 1/1 mice. Western Blot analysis revealed that HCN1 protein expression in neonatal cortical plate tissue of HCN 1/1 mice amounted to only 3% of the HCN1 in young adult cortex and suggested that in HCN1 2/2 mice other isoforms (particularly HCN4) might be compensa-tory up-regulated. At the first day after birth, functional ablation of the HCN1 subunit did not affect the proportion of I h expressing pyramidal cortical plate neurons. Although the contribution of individual subunit proteins remains open, the lack of HCN1 markedly slowed the current activation and deactivation in individual I h expressing neurons. However, it did not impair maximal amplitude/density, voltage dependence of activation, and cAMP sensitivity. In conclusion, our data imply that, although expression is relatively low, HCN1 contributes substantially to I h properties in individual cortical plate neurons. These properties are significantly changed in HCN1 2/2 , either due to the lack of HCN1 itself or due to compensatory mechanisms.
Background/Aims: Hyperpolarization activated cyclic nucleotide gated 1 (HCN1) channels determine ... more Background/Aims: Hyperpolarization activated cyclic nucleotide gated 1 (HCN1) channels determine neuronal excitability in several brain regions. In contrast to HCN2 and HCN4, HCN1 is less sensitive to cAMP and the number of other known modulators is limited. One of those, the protein kinase C (PKC), showed opposing effects on mouse HCN1 channels expressed in Xenopus oocytes. Methods: In order to study PKC effects on HCN1 mediated currents in a mammalian environment we expressed rat HCN1 or human HCN1 in human embryonic kidney (HEK293) cells and rat HCN1 in murine neuroblastoma (N1E-115) cells. We recorded the resulting I h before and during the application of the membrane permeable nonmetabolizable PKC-activator 4βPMA in cell-attached mode of the patch-clamp technique, leaving the intracellular environment intact. Results: 4βPMA reduced maximal HCN1 mediated currents to about 60-70 % and slowed its activation, but left its voltage sensitivity unchanged. The effect was neither due to species-related differences nor restricted to HEK293 cells, because it was comparable for human and rat HCN1 in HEK293 and for rat HCN1 in N1E-115 cells. However, pre-treatment with the PKC blocker GF109203X abolished 4βPMA induced I h changes. Disrupting the intracellular environment by recording in whole-cell mode drastically reduced the 4βPMA effect. Conclusion: PKC activation reduces and slows I h in non-neuronal and neuronal mammalian cells transfected with rat or human HCN1 if the intracellular content remains intact. 535 Reetz/Strauss: PKC Mediated HCN1 Inhibition Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry P = 0.3; /F under 1 µM 4βPMA or V 1/2ctrl = -98.0 ± 3.0 mV vs. V 1/24βPMA = -101.9 ± 1.7 mV n = 8, paired t-test: P = 0.07 under 0.2 µM 4βPMA). These data suggest that activation of PKC results in a suppression of maximal rHCN1 mediated current accompanied by a slowing of its activation.
Quantitative trait loci (QTL) analysis has been carried out to identify genes controlling aluminu... more Quantitative trait loci (QTL) analysis has been carried out to identify genes controlling aluminum tolerance in rice. One hundred and six doubled haploid plants derived from a cross between a japonica variety, Azucena, and an indica variety, IR64, were used for QTL mapping and 175 RFLP and isozyme markers were employed to identify QTLs. QTL analysis revealed the presence of two QTLs, QTL 1 and QTL 2, which located in the middle of chromosome 6 and at the end of chromosome 8, respectively. The two QTLs explained 21.6% of the total phenotypic variation in the population based on a multiple regression model.
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 ×
At present, the most efficient therapeutical treatment of multiple sclerosis (MS) is achieved by ... more At present, the most efficient therapeutical treatment of multiple sclerosis (MS) is achieved by IFN-ß. However, its in vivo effects remain incompletely understood. If applied parenterally, the hydrophobic IFN-ß acts primarilyon blood cells with probable selectivity tor functionally different lymphocyte subpopulations, monocytes and granulocytes. We have investi!ated the expression of the activation marker interleukin-2 receptor-a (CD25) on CD3+ T cells, CD19 B cells, foetal-type 'Y8+CD3+ T cells and foetal-type CD5+CD19+ B cells of the peripheral blood. In addition, the oxidative burst activity and apoptosis have been determined in mononuclear and polymorphonuclear blood cells, respectively. The study accompanied aphase rn trial with IFN-ßlb (BETAFERON~, Schering). Two groups ofMS patients with relapsing-remitting course of the disease have been investigated at 8 time points (days 0, 5, 15,31,60,90, 180 and 270 after starting therapy): (1) verum group (n = 8) with application of8 Mill. units IFN-ßlb every other day, and (2) placebo group (n = 4) with application ofplacebo fOT 3 months and therapy as in (1) from day 90 onward. The main results were: (1) Activated T cells decreased until day 180 in the verum group and return thereafter to pre-treatment values, whereas in the placebo group the values remained relatively stahle over the whole observation period. (2) Activated B cells increased between days 90 and 270 in both groups, i.e. after verum application in both groups. (3) Foetal-type B cells were more activated than total B and T cells with increase over time in both groups.
Neuronal progenitor cells delivering neurotrophic factors are a promising therapeutic tool for tr... more Neuronal progenitor cells delivering neurotrophic factors are a promising therapeutic tool for treatment of neurodegenerative diseases. Although several promising results have come from studies in different animal models, detailed knowledge of the action of neurotrophic factors in the CNS is still lacking. A clonally derived, immortalized rat striatal cell line (ST14A) expressing ciliary neurotrophic factor (CNTF) offers a stable and controlled background with which to analyze CNTF actions on the transcriptional level in CNS progenitor cells. To identify early transcriptional changes induced by CNTF expression, we transfected the CNTF gene into ST14A cells, which differentiate at the nonpermissive temperature of 39°C via suppression of the immortalizing SV40 large T antigen. This shows a CNTF-dependent hypoxic/ ischemic stress response during the earliest stage of differentiation, with expression of specific transcripts and evidence of translational repression leading to decreased protein synthesis in the transfected cells. This process is mediated by the Ras/MAP kinase pathway and is accompanied by impaired proliferation and metabolism as well as signs of neuronal differentiation. The stress-like response in the early stage of differentiation improves the ability of the transfected cells to respond to and cope with a stressful environment in vivo. The present data indicate higher viability, longer life, and greater differentiation capacity of CNTF-ST14A cells if they are used for transplantation. We conclude that the stress-like response during the early stage of differentiation improves the ability of the CNTF-ST14A cells to respond and adapt to a stressful environment, which renders them useful candidate cells for in vivo trials of treatment for neurodegenerative diseases in animal models, e.g., of Huntington's disease.
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.
Deep brain stimulation (DBS) has become a treatment for a growing number of neurological and psyc... more Deep brain stimulation (DBS) has become a treatment for a growing number of neurological and psychiatric disorders, especially for therapy-refractory Parkinson's disease (PD). However, not all of the symptoms of PD are sufficiently improved in all patients, and side effects may occur. Further progress depends on a deeper insight into the mechanisms of action of DBS in the context of disturbed brain circuits. For this, optimized animal models have to be developed. We review not only charge transfer mechanisms at the electrode/tissue interface and strategies to increase the stimulation's energy-efficiency but also the electrochemical, electrophysiological, biochemical and functional effects of DBS. We introduce a hemi-Parkinsonian rat model for long-term experiments with chronically instrumented rats carrying a backpack stimulator and implanted platinum/iridium electrodes. This model is suitable for (1) elucidating the electrochemical processes at the electrode/tissue interface, (2) analyzing the molecular, cellular and behavioral stimulation effects, (3) testing new target regions for DBS, (4) screening for potential neuroprotective DBS effects, and (5) improving the efficacy and safety of the method. An outlook is given on further developments of experimental DBS, including the use of transgenic animals and the testing of closed-loop systems for the direct on-demand application of electric stimulation.
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.
In rat models of Parkinson's disease, injections of 6-hydroxydopamine (6-OHDA) into different are... more In rat models of Parkinson's disease, injections of 6-hydroxydopamine (6-OHDA) into different areas of the basal ganglia result in dopaminergic neurodegeneration in the substantia nigra. The extent and time course of the dopaminergic lesions varies between the models. While the effects on neurons have been extensively studied, little is known about the effects on astrocytes. We compared astrocytic activation (i.e. increase in number and staining intensity of glial fibrillary acidic protein immunoreactive cells) at the injection site and in downstream structures of the motor loop, i.e. the globus pallidus (GP) and the subthalamic nucleus (STN) following 6-OHDA lesion of the medial forebrain bundle (MFB) or the striatum. Lesions in both regions resulted in astrocytic activation at the lesion site, but their remote effects varied. MFB injections caused astrocytic activation in the ipsi-and contralateral striatum, whereas striatal injections resulted in astrocytic activation in the GP and STN. Since 6-OHDA injections into the MFB and the striatum result in complete and partial SNc lesions, respectively, we hypothesize that communication links exist between astrocytes, or between neurons and astrocytes, along neuronal pathways that transmit activating signals in response to neuronal damage-but only if the neuronal pathways are at least partially intact. ß
Apart from a central function in the extrapyramidal motor system, dopamine has been suggested to ... more Apart from a central function in the extrapyramidal motor system, dopamine has been suggested to play a role in neuroimmune interactions. Particularly in diseases of the central nervous system, such as multiple sclerosis, alterations in dopamine homeostasis might have immunological consequences. We investigated potential effects of dopamine stabilized by ascorbic acid on specifically activated encephalitogenic T cells at the peak of activation. Those cells exhibited an upregulation of voltage-sensitive K + channels which play a role in many neurotransmitter responses of lymphocytes and fulfilled a prerequisite to respond to dopamine, i.e. stable expression of mRNA for dopamine receptors DRD1, DRD2 and DRD3. However, whole-cell and perforated whole-cell recordings revealed no change in voltage-sensitive K + currents. Moreover, T cell proliferation was not changed in the presence of dopamine. Previously reported dopamine effects on T cells may be explained by a comparatively lower activation of the cells under investigation, suggesting an activation dependence of dopamine effects that may not be mediated by K + channels. Alternatively, the occurrence of dopamine degradation products under unprotected conditions may account for the changes reported. Nevertheless, care should be taken when using the dopamine-protecting anti-oxidant ascorbic acid, since we found that it markedly inhibited both K + currents and lymphocyte proliferation at higher concentrations.
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