Interaction of androsterone and progesterone with inhibitory ligand-gated ion channels: a patch clamp study.

Gamma-aminobutyric acid receptor type A (GABA(A)) receptor channels mediate fast inhibitory neurotransmission throughout the central nervous system while the expression of ionotropic glycine receptors is mainly restricted to the spinal cord and brain stem. Neuroactive steroids are well known as positive allosteric modulators of GABA(A) receptor function. Furthermore, there have been hints for an interaction of neuroactive steroids with ionotropic glycine receptors. The aim of the study was to characterize the effect of androsterone and progesterone on alpha(1) and alpha(1)beta glycine receptor and alpha(1)beta(2)gamma(2) GABA(A) receptor channels and to examine the molecular interactions between ligands and receptors. Electrophysiological recordings were performed on HEK 293 cells using the patch clamp technique in combination with an ultrafast perfusion system. A direct activation of inhibitory ionotropic receptors was observed for androsterone at GABA(A) receptor channels. A coactivation of currents elicited by nonsaturating agonist concentrations was observed with androsterone and progesterone at glycine and GABA(A) receptor channels. We could show that association of beta subunits with alpha subunits affects the sensitivity of glycine receptors to androsterone. In contrast to previous reports in which recombinant glycine receptors were inhibited by progesterone, a potentiating effect was revealed by our experiments. At concentrations of 0.1 mM and higher, there were also hints to a channel block-like mechanism. In conclusion, different molecular mechanisms of interaction between neuroactive steroids and GABA as well as glycine receptors could be identified and quantitatively described. Our data clarify the role of steroid compounds in the modulation of inhibitory receptor channel function.

Molecular mechanisms of interaction between the neuroprotective substance riluzole and GABA(A)-receptors.

The antiepileptic drug riluzole is used as a therapeutic agent in amyotrophic lateral sclerosis due to its neuroprotective effects. Besides presynaptic inhibition of GABAergic and preferentially glutamatergic transmission, it also potentiates postsynaptic GABA(A)-receptor function. We investigated the postsynaptic effects of riluzole on GABA(A)-receptor channels by use of the patch-clamp technique. Recombinant alpha1beta2gamma(2s) and alpha1beta2 GABA(A) receptors were expressed in HEK 293 cells by transient transfection. Pulses of GABA were applied in combination with different concentrations of riluzole to whole cell or outside-out patches with either alpha1beta2gamma(2s) or alpha1beta2 GABA(A)-receptor channels. Co-application of riluzole led to a slight decrease of absolute peak current amplitudes and steady-state currents in prolonged presence of GABA at saturating concentrations. In the presence of riluzole, enhancement of current amplitudes was observed with lower concentrations of GABA at alpha1beta2gamma(2s) receptors and to a lower extent also at alpha1beta2 receptors. Thus, the potentiating effect of riluzole was shown to be not abolished in the absence of the gamma(2s)-subunit. A further prominent effect of riluzole was a highly significant acceleration of the time course of current decay, most probably pointing to an open-channel block-like mechanism of action. As both receptor subtypes were affected similarly by the block, it could be concluded that the respective binding sites should be assumed within a region of high sequence homology like it is given for the channel-lining M2 domain of GABA(A)-receptor subunits. In conclusion, three different molecular mechanisms of interaction of the neuroprotective compound riluzole were observed at two different subtypes of GABA(A) receptor channels. The results further point to the impact of the inhibitory as well as the excitatory synaptic activity as a pharmacological target to counteract chronic excitotoxicity and reveal molecular mechanisms of action of the only one neuroprotective drug in current clinical use in patients suffering from amyotrophic lateral sclerosis.

Role of endotoxin in the pathogenesis of critical illness polyneuropathy.

Critical illness polyneuropathy (CIP) occurs in association with sepsis and multiple organ failure; however, little is known about the pathomechanisms of CIP and its therapy. In order to determine the parameters which interfere with development of CIP, electrophysiological investigations of peripheral nerves and biochemical measures were correlated to each other. The present study includes 20 consecutive patients in an intensive care unit developing severe sepsis or septic shock. Nerve conduction studies and electromyography were performed with occurring sepsis (day 1, 7, 14) and neurophysiological parameters were correlated with biochemical measures, especially indicators of infection and inflammation. It was found that all patients developed neurophysiological signs of axonal motor polyneuropathy. There was a significant correlation between serum concentrations of endotoxin and interleukin-2 receptors (IL2-R) and reduction of the amplitude of the compound motor action potentials. Other clinical and biochemical parameters showed no significant correlations with neurophysiological data. This finding apparently indicates that endotoxin damages nerve axons directly or indirectly, e.g. by activation of inflammatory cascades (IL2-R). Endotoxin appears to be an essential factor in the pathogenesis of CIP in sepsis, and therapeutic options neutralizing endotoxin may prevent development of CIP.

Temporospatial coupling of networked synaptic activation of AMPA-type glutamate receptor channels and calcium transients in cultured motoneurons.

AMPA-type glutamate receptor (GluR) channels provide fast excitatory synaptic transmission in the CNS, but mediate also cytotoxic insults. It could be shown that AMPA-type GluR channel-mediated chronic excitotoxicity leads to an increased intracellular calcium concentration and plays an important role in neurodegenerative diseases like for example amyotrophic lateral sclerosis (ALS). As calcium is an important mediator of various processes in the cell and calcium signals have to be very precise in the temporospatial resolution, excessive intracellular calcium increases can seriously impair cell function. It is still unclear if AMPA-type receptors can directly interact with the intracellular calcium homeostasis or if other mechanisms are involved in this process. The objective of this study was therefore to investigate the calcium homeostasis in rat motoneurons under physiological stimulation of AMPA-type GluR channels using calcium imaging techniques and patch-clamp recordings simultaneously. It was found that spontaneous excitatory postsynaptic currents of cultured motoneurons did not elicit significant intracellular calcium transients. Large intracellular calcium transients occurred only when preceding fast sodium currents were observed. Pharmacological experiments showed that activation of AMPA-type GluR channels during synaptic transmission has a great functional impact on the calcium homeostasis in motoneurons as all kinds of activity was completely blocked by application of the selective kainate- and AMPA-type GluR channel blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Furthermore we suggest from our experiments that calcium transients of several hundred milliseconds' duration result from release of calcium from the endoplasmic reticulum via activation of ryanodine receptors (calcium-induced calcium release, CICR). Our results help to understand the regulatory function of AMPA-type GluR channels in the intracellular calcium homeostasis which is known to be disturbed in neurodegenerative diseases.

High-affinity blockade of voltage-operated skeletal muscle sodium channels by 2,6-dimethyl-4-chlorophenol.

BACKGROUND AND OBJECTIVE: The aromatic alcohol most closely resembling the aromatic tail of lidocaine is 2,6-dimethylphenol. This agent is as potent as lidocaine in blocking voltage-operated sodium channels. The aim of this study was to show the effect of halogenation in the para-position on the potency of this compound to block voltage-operated sodium channels. METHODS: Insertion of the halogen chloride into the para-position of the molecule 2,6-dimethylphenol yielded 2,6-dimethyl-4-chlorophenol. Block of sodium currents by this compound was studied using heterologously expressed voltage-operated rat neuronal (rat IIa) sodium channels. RESULTS: 2,6-dimethyl-4-chlorophenol reversibly suppressed depolarization-induced whole-cell sodium inward currents. The ECR50 for block of resting channels at a hyperpolarized holding potential (-150 mV) was 127 micromol, the Hill coefficient nH 1.7. Membrane depolarization inducing either fast or slow-inactivation strongly increased the blocking potency. This is an important feature of a local-anaesthetic-like action. The estimated half-maximum effect concentration for the fast-inactivated channel state ECI50 was 28 micromol, the Hill coefficient nH 3.8. When 20-30% of channels were slow-inactivated using long (2.5 s) prepulses, followed by a 10 ms repolarization period to allow recovery from fast inactivation, the IC50 at -100 mV holding potential was reduced to 53 micromol. CONCLUSION: These results, which show that 2,6-dimethyl-4-chlorophenol blocks voltage-operated sodium channels in a lidocaine-like manner while having a several fold higher potency than the non-halogenated parent compound, highlight a potentially meaningful principle of increasing the sodium channel blocking potency of phenol derivatives.

The cellular mRNA expression of GABA and glutamate receptors in spinal motor neurons of SOD1 mice.

ALS is a fatal neurodegenerative disorder characterized by a selective loss of upper motor neurons in the motor cortex and lower motor neurons in the brain stem and spinal cord. About 10% of ALS cases are familial, in 10-20% of these, mutations in the gene coding for superoxide dismutase 1 (SOD1) can be detected. Overexpression of mutated SOD1 in mice created animal models which clinically resemble ALS. Abnormalities in glutamatergic and GABAergic neurotransmission presumably contribute to the selective motor neuron damage in ALS. By in situ hybridization histochemistry (ISH), we investigated the spinal mRNA expression of the GABAA and AMPA type glutamate receptor subunits at different disease stages on spinal cord sections of mutant SOD1 mice and control animals overexpressing wild-type SOD1 aged 40, 80, 120 days and at disease end-stage, i.e. around 140 days) (n=5, respectively). We detected a slight but statistically significant decrease of the AMPA receptor subunits GluR3 and GluR4 only in end stage disease animals.

Ligand-gated channels in early mesencephalic neuronal precursors: immunocytochemical and electrophysiological analysis.

Neuronal precursors play an important role in potential regenerative therapeutic strategies in different neurodegenerative diseases, e.g. Parkinson's disease. To understand proliferation and differentiation of these cells in vitro and in vivo, it is important to characterize functional properties of neuronal precursors in detail. The aim of the present study was to analyse the electrophysiological characteristics of ligand-gated channels of neuronal precursors prepared from the rat ventral mesencephalon (VM) of embryonic stage 12.5 during their in vitro differentiation. For the experiments we used the patch-clamp technique in combination with a system for ultrafast solution exchange and immunocytochemistry. It could be shown that functional active AMPA-type glutamate as well as GABA(A) receptor channels are expressed at an early stage of neuronal development. In culture we observed excitatory as well as inhibitory postsynaptic currents (defined by their different kinetics) which correspond to the activation of AMPAergic and GABAergic receptor channels. Two populations of glutamate-activated currents could be differentiated by their different time course of desensitization whereas the time course of resensitization and deactivation was normally distributed in all cells. GABAergic currents could be blocked by bicuculline and their kinetics correspond to that of GABA(A) receptor channel currents. Summarizing the results, in the present study it was shown for the first time that neuronal embryonic precursors of the rat VM express both functional AMPA-type glutamate and functional GABA(A) receptor channels in vitro.

Intrathecal synthesis of monocyte chemoattractant protein-1 (MCP-1) in amyotrophic lateral sclerosis: further evidence for microglial activation in neurodegeneration.

Autopsy studies and animal experiments suggest that microglial inflammation contributes to the pathogenesis of amyotrophic lateral sclerosis (ALS). Monocyte-chemoattractant protein (MCP-1) might play an important role in microglial recruitment. We studied MCP-1 levels in sera and cerebrospinal fluid of 29 ALS patients and compared the results with 11 control patients with tension headache. The MCP-1 level was determined using enzyme-linked immunosorbent assays (ELISA). A significant increase in cerebrospinal fluid MCP-1 level but not serum level was seen in the patients with ALS compared to the control subjects. These results suggest that cerebrospinal fluid MCP-1 activity may be a sensitive marker for neuroinflammation in ALS useful for monitoring treatment trials in ALS.

Kinetic analysis of recombinant mammalian alpha(1) and alpha(1)beta glycine receptor channels.

To analyze the influence of the beta-subunit on the kinetic properties of GlyR channel currents, alpha(1)-subunits and alpha(1)beta-subunits were transiently expressed in HEK 293 cells. A piezo dimorph was used for fast application of glycine to outside-out patches. The rise time of activation was dose dependent for both receptors and decreased with increasing glycine concentrations. Subunit composition had no effect on the time course of activation. Coexpression of alpha(1)- and beta-subunits resulted in a significantly lower EC(50) and a reduced slope of the dose-response curve of glycine compared with expression of alpha(1)-subunits alone. For both receptor subtypes, the time course of desensitization was concentration dependent. Desensitization was best fitted with a single time constant at 10-30 micro M, with two at 0.1 mM, and at saturating concentrations (0.3-3 mM) with three time constants. Desensitization of homomeric alpha(1)-receptor channels was significantly slower than that of alpha(1)beta-receptor channels. The time course of current decay after the end of glycine pulses was tested at different pulse durations of 1 mM glycine. It was best fitted with two time constants for both alpha(1) and alpha(1)beta GlyR channels, and increased significantly with increasing pulse duration.

Rapsyn N88K is a frequent cause of congenital myasthenic syndromes in European patients.

BACKGROUND: Mutations in various genes of the neuromuscular junction may cause congenital myasthenic syndromes (CMS). Most mutations identified to date affect the epsilon-subunit gene of the acetylcholine receptor (AChR), leading to end-plate AChR deficiency. Recently, three different mutations in the RAPSN gene have been identified in four CMS patients with AChR deficiency. OBJECTIVE: To perform mutation analysis of the RAPSN gene in patients with sporadic or autosomal recessive CMS. METHODS: One hundred twenty CMS patients from 110 unrelated families were analyzed for the RAPSN mutation N88K by restriction fragment length polymorphism and sequence analysis. RESULTS: In 12 CMS patients from 10 independent families, RAPSN N88K was identified either homozygous or heteroallelic to another missense mutation. Symptoms usually started perinatally or in the first years of life. However, one patient did not show any myasthenic symptoms before the third decade. Clinical symptoms typically included bilateral ptosis, weakness of facial, bulbar, and limb muscles, and a favorable response to anticholinesterase treatment. Crisis-like exacerbations with respiratory insufficiency provoked by stress, fever, or infections in early childhood were frequent. All RAPSN N88K families originate from Central or Western European countries. Genotype analysis indicated that they derive from a common ancestor (founder). CONCLUSIONS: The RAPSN mutation N88K is a frequent cause of rapsyn-related CMS in European patients. In general, patients (RAPSN N88K) were characterized by mild to moderate myasthenic symptoms with favorable response to anticholinesterase treatment. However, severity and onset of symptoms may vary to a great extent.

IgG from patients with Guillain-Barré syndrome interact with nicotinic acetylcholine receptor channels.

In Guillain-Barré syndrome (GBS), immunoglobulin G (IgG) antibodies block neuromuscular transmission pre- and postsynaptically and thus are of potential pathogenic relevance. We investigated whether IgG from GBS patients has a direct interaction with nicotinic acetylcholine receptor (nAChR) channels. Purified IgG fractions from six GBS patients that blocked neuromuscular transmission in a previous study were analyzed by the patch-clamp technique in combination with an ultrafast system for solution exchange. Sera from three patients with other inflammatory neurological disorders were used as controls. Mouse myotubes expressing native embryonic-type nAChR channels and human embryonic kidney (HEK) 293 cells transiently transfected with recombinant adult-type nAChR channels were used. Repeated 20-ms pulses of acetylcholine (ACh) were applied to outside-out patches in the presence of GBS-IgG. IgG of the patients had a significant reversible blocking action on embryonic- and adult-type nAChR channels with some variability in the magnitude of the block. Activation and desensitization kinetics were not affected when GBS-IgG was applied. None of the control sera blocked the AChR channels. The observed postsynaptic block effect fulfills the criteria of a channel-blocking IgG antibody similar to those seen in autoimmune myasthenia and may contribute to muscle weakness during the acute phase of GBS.

Analysis of a slow desensitized state of recombinant adult-type nicotinic acetylcholine receptor channels.

A characteristic feature of the kinetics of nicotinic acetylcholine receptor (nAChR) channels is fast and nearly complete desensitization with a time course between 10 and 100 ms and recovery from desensitization in the range of some hundred ms. In the present study we used a piezo-driven system for ultra-fast solution exchange, analysed the recovery from the fast desensitized state of mouse recombinant adult-type nAChR channels and found no difference to that of embryonic-type channels. By double pulse experiments with application of pulses with a saturating concentration of 1 mm acetylcholine (ACh) with increasing duration of the first pulse and a constant interval between pulses we detected a second slow desensitized state which was entered with a time constant of 2835 ms. Recovery from the slow desensitized state proceeded with a single exponential with a time constant of 16134 ms. The experimental data were interpreted by the addition of a transition from the desensitized state with two bound ACh molecules to a slow desensitized state to the well known circular kinetic scheme of activation and desensitization of nAChR channels. This slow desensitized state might play a role in muscle fatigue or in pathological states like myasthenic syndromes.

[Cerebellar symptoms in motor neuron diseases. Special form of amyotrophic lateral sclerosis plus syndrome]. / Zerebelläre Symptome bei Motoneuronerkrankungen. Spezielle Form des ALS-Plus-Syndroms.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive and selective loss of motor neurons in the cerebral cortex, brainstem, and spinal cord. The upper motor neuron syndrome is characterized by symptoms of spastic paresis. Muscle weakness and atrophy, fasciculations, and cramps are typical signs for the degeneration of the lower motor neurons. In 1994, the El Escorial criteria were proposed for the diagnosis of ALS. These criteria include ALS-plus syndromes, which are defined by an association of ALS with extrapyramidal features or dementia. In this paper, we present two cases of ALS associated with signs of cerebellar degeneration. According to the revised El Escorial criteria, the described unusual combination of upper and lower motor neuron signs in association with cerebellar ataxia can be classified as a specific form of ALS-plus syndromes.

Voltage-dependent block of neuronal and skeletal muscle sodium channels by thymol and menthol.

BACKGROUND AND OBJECTIVE: Thymol is a naturally occurring phenol derivative used in anaesthetic practice as a stabilizer and preservative of halothane, usually at a concentration of 0.01%. Although analgesic effects have long been described for thymol and its structural homologue menthol, a molecular basis for these effects is still lacking. We studied the blocking effects of thymol and menthol on voltage-activated sodium currents in vitro as possible molecular target sites. METHODS: Whole cell sodium inward currents via heterologously (HEK293 cells) expressed rat neuronal (rat type IIA) and human skeletal muscle (hSkM1) sodium channels were recorded in the absence and presence of definite concentrations of either thymol or menthol. RESULTS: When depolarizing pulses to 0 mV were started from a holding potential of -70 mV, half-maximum blocking concentrations (IC50) for the skeletal muscle and the neuronal sodium channel were 104 and 149 mumol for thymol and 376 and 571 mumol for menthol. The blocking potency of both compounds increased at depolarized holding potentials with the fraction of inactivated channels. The estimated dissociation constant Kd for thymol and menthol from the inactivated state was 22 and 106 mumol for the neuronal and 23 and 97 mumol for the skeletal muscle sodium channel, respectively. CONCLUSIONS: The results suggest that antinociceptive and local anaesthetic effects of thymol and menthol might be mediated via blockade of voltage-operated sodium channels with the phenol derivative thymol being as potent as the local anaesthetic lidocaine.

[Primary angiitis of CNS (PACNS)]. / Die primäre Angiitis des ZNS (PACNS).

Primary angiitis of the CNS (PACNS) is a rare inflammatory disease affecting middle-aged patients. The angiitis is focal and segmental in distribution, involving small and medium-sized leptomeningeal and intracranial vessels. The most frequent presenting symptoms are headaches, focal neurologic deficits, and confusion. In addition, aphasia, neuropsychological deficits, and seizures may occur. The paper presents a review of the literature. In addition, a typical case of PACNS is presented. This case demonstrates that diagnosis of PACNS is difficult and brain biopsy is essential to confirm it.

Functional diversity of recombinant human AMPA type glutamate receptors: possible implications for selective vulnerability of motor neurons.

Lower motor neurons are known to be susceptible to glutamate-mediated cell damage via overstimulation of AMPA type glutamate receptors (GluR). The molecular basis of an important hypothesis in investigating amyotrophic lateral sclerosis (ALS) is glutamate-excitotoxicity. The aim of this study was to define desensitization and deactivation kinetics of recombinant human GluR1 and GluR2 receptor channels and their splice variants by means of patch-clamp experiments employing ultrafast solution exchange techniques. By this approach, the desensitization time constants of homooligomeric channels could be measured as tau(Des)=2.95+/-0.22 ms (n=10) for GluR1flip, tau(Des)=3.17+/-0.19 ms (n=10) for GluR1flop, tau(Des)=9.86+/-0.79 ms (n=10) for GluR2flip, and tau(Des)=1.87+/-0.26 ms (n=10) for GluR2flop, respectively. In the case of GluR1flip/flop and GluR2flop, a nondesensitising steady state current of less than 1% of peak current amplitude was observed, while GluR2flip channel currents showed a marked steady state component of about 10% of the maximum current. No significant differences were detected comparing the deactivation time course of GluR1 and GluR2 splice variants. These results suggest that the human GluR subtypes tested comprise no fundamental difference to their rodent analogous. Therefore, we describe a preparation that will be useful for further investigation of motor neuron physiological properties and a methodological approach allowing to study functional recombinant human GluR channels under reliable conditions.

Pentobarbital and brilliant green modulate the current response of recombinant rat kainate-type GluR6 receptor channels differentially.

Kainate-type receptor channels (GluR5-7, KA1,2) belong to the family of ionotropic glutamate receptor channels. In the present study we tested the interaction of two different drugs with GluR6 channels using outside-out patches from HEK cells transiently transfected with cDNA of GluR6 channels. Glutamate and the respective drugs were delivered by a system for ultrafast solution exchange. Application of a saturating concentration of 3 mM glutamate resulted in fast current transients with desensitization time constants between 3 and 10 ms. Addition of pentobarbital (>or=1 mM) to the 3 mM glutamate containing test-solution resulted in a significant decrease of the time constant of current decay without affecting the peak current amplitude. Brilliant green (>or=1 mM) had the opposite effect and led to an increase of the time constant of current decay after application of 3 mM glutamate. The pharmacological effects of both drugs were completely reversible. Additionally, a significant increase of the peak current amplitude and the time constant of deactivation in presence of brilliant green was observed. Summarizing our results, we could identify a further substance, brilliant green, interacting with GluR6 kainate-type receptor channels.

Deactivation and desensitization of mouse embryonic- and adult-type nicotinic receptor channel currents.

Recombinant nicotinic acetylcholine receptor (nAChR) channels transiently expressed in HEK293 cells were investigated using the patch-clamp technique in the cell-attached and outside-out modes for single-channel analysis and ultra-fast agonist application to multiple channels. Deactivation (current decay after removal of agonist) and desensitization (current decay in the presence of agonist) were analyzed at embryonic- (gamma) and adult-type (epsilon) nAChR channels. Time constants of desensitization were similar for both receptor types (epsilon: 53.1+/-16.9 ms; gamma: 49.2+/-15.7 ms) and corresponded to the mean duration of clusters of single channel openings activated by pulses of 1 mM ACh. Deactivation showed distinct characteristics. Time constants were 1.76+/-0.16 ms for epsilon- and 3.19+/-0.18 ms for gamma-nAChR channels, corresponding to mean burst duration analyzed from single channels in the same preparation (epsilon: 1.85+/-1.2 ms, gamma: 3.85+/-2.1 ms). It is assumed that differences in deactivation are of functional relevance at the muscle endplate.