Karl J. Föhr

Increased calcium entry into dystrophin‐deficient muscle fibres of MDX and ADR‐MDX mice is reduced by ion channel blockers

1 Single fibres were enzymatically isolated from interosseus muscles of dystrophic MDX mice, myotonic‐dystrophic double mutant ADR‐MDX mice and C57BL/10 controls. The fibres were kept in cell culture for up to 2 weeks for the study of Ca2+ homeostasis and sarcolemmal Ca2+ permeability. 2 Resting levels of intracellular free Ca2+, determined with the fluorescent Ca2+ indicator fura‐2, were slightly higher in MDX (63 ± 20 nm; means ±s.d.; n= 454 analysed fibres) and ADR‐MDX (65 ± 12 nm; n= 87) fibres than in controls (51 ± 20 nm; n= 265). 3 The amplitudes of electrically induced Ca2+ transients did not differ between MDX fibres and controls. Decay time constants of Ca2+ transients ranged between 10 and 55 ms in both genotypes. In 50% of MDX fibres (n= 68), but in only 20% of controls (n= 54), the decay time constants were > 35 ms. 4 Bath application of Mn2+ resulted in a progressive quench of fura‐2 fluorescence emitted from the fibres. The quench rate was about 2 times higher in MDX fibres (3.98 ± 1.9% min−1; n= 275) than in controls (2.03 ± 1.4% min−1; n= 204). The quench rate in ADR‐MDX fibres (2.49 ± 1.4% min−1; n= 87) was closer to that of controls. 5 The Mn2+ influx into MDX fibres was reduced to 10% by Gd3+, to 19% by La3+ and to 47% by Ni2+ (all at 50 μm). Bath application of 50 μm amiloride inhibited the Mn2+ influx to 37%. 6 We conclude that in isolated, resting MDX muscle fibres the membrane permeability for divalent cations is increased. The presumed additional influx of Ca2+ occurs through ion channels, but is well compensated for by effective cellular Ca2+ transport systems. The milder dystrophic phenotype of ADR‐MDX mice is correlated with a smaller increase of their sarcolemmal Ca2+ permeability.

Atomoxetine acts as an NMDA receptor blocker in clinically relevant concentrations

Background and purpose:  There is increasing evidence that not only the monoaminergic but also the glutamatergic system is involved in the pathophysiology of attention‐deficit hyperactivity disorder (ADHD). Hyperactivity of glutamate metabolism might be causally related to a hypoactive state in the dopaminergic system. Atomoxetine, a selective noradrenaline reuptake inhibitor, is the first non‐stimulant approved for the treatment of this disorder. Here we have evaluated the effects of atomoxetine on glutamate receptors in vitro.

Classical and Nonclassical Estrogen Action in the Developing Midbrain

There is widespread acceptance that estrogen is involved in various steps of cellular differentiation during brain development. In the past years, we have demonstrated such a developmental role for estrogen in the rodent midbrain. Precisely, estrogen affects midbrain dopamine neurons with respect to functional and morphological maturation. On the cellular level, estrogen may act classically by binding and activating its respective nuclear receptors, thereby controlling the transcription of target genes. On the other hand, many estrogen effects in the CNS are transmitted nonclassically by interactions with putative membrane receptors and by stimulating distinct intracellular signaling cascades. In the midbrain, classical and nonclassical estrogen signaling routes operate side by side to ensure the proper development of dopaminergic cells. In the present report, we detail some of the cellular and molecular events which are activated by estrogen and are thought to take part in the estrogen-mediated stimulation of dopamine neuron differentiation.

Developmental and Functional Nature of Human iPSC Derived Motoneurons

One of the major functional properties of the mature motoneuron is its ability to generate and conduct signals from the central nervous system (CNS) to the peripheral muscle cell in order to induce and control muscle contraction [1]. The molecular composition of the neuromuscular junction (NMj) is crucial for its function and maintenance whereas dysregulation of endplate physiology is considered to be involved in denervation of the muscle cells and subsequent motoneuron degeneration [2, 3]. At early developmental stages of the neuron-to-muscle synaptogenesis, a large number of spinal motoneurons die, presumably because they fail to form adequate connections with the target muscle. In fact, if the limb bud (the precursor of limb muscles) is removed before the formation of neuromuscular connections all the corresponding motoneurons eventually degenerate [4]. In vitro, various cell systems are utilized to search for developmental and functional characteristics of the motoneuron system. Both, primary cultures and stem cellderived motoneurons are used for various questions. Pluripotent embryonic stem cells [5] from mouse and human origin [6] had been shown to be able to generate motoneurons in vitro. Since the first discovery and invention of the induced pluripotent stem cell (iPSC) technology by Takahashi and Yamanaka [7], it is now possible to analyze and study cell development and differentiation on the basis of a gene defect in patient specific settings [8, 9]. For comparison all these studies are crucially dependent upon the analysis of human cell differentiation, morphology and protein expression under

Rapid increase of glial glutamate uptake via blockade of the protein kinase A pathway

Glutamate is the main excitatory neurotransmitter in the vertebrate central nervous system. Removal of this transmitter from the synaptic cleft by glial and neuronal transporter systems plays an important role in terminating glutamatergicneurotransmission. The effects of different activators and blockers of PKA and PKC on glutamate uptake were studied in primary glial cells cultivated from the rat cortex using the patch‐clamp recording technique and immunocytochemical methods. GF 109203X enhances glutamate‐induced membrane currents in a concentration‐ and time‐dependent manner. After pre‐application for 40 s the maximal transport capacity was increased by 30–80%. The estimated Km‐value of the transport system did not change after drug application and the enhanced glutamate uptake was reversible within a few minutes upon washout. Activators and blockers of the PKC pathway did not affect glutamate uptake, whereas H89, a selective blocker of PKA, mimicked the effects of GF 109203X, indicating involvement of the protein kinase A pathway. The GF 109203X‐induced increase in transport capacity is likely to be mediated by GLAST since the GLT‐1 selective blocker dihydrokainate was unable to block basal or stimulated glutamate uptake. Furthermore, the increase in transport activity may well be based on an increase in cell surface expression of the transporter protein since preincubation with cytochalasin‐B, a protein that blocks actin polymerization, almost completely abolished the effect of GF 109203X and H89. These results indicate that GF 109203X and H89 enhance glial glutamate uptake via blockade of the PKA. The described effect may affect glutamatergic neurotransmission by reducing the glutamate concentration in the synaptic cleft.

atomoxetine affects transcription/translation of the nMDa receptor and the norepinephrine transporter in the rat brain - an in vivo study

Attention-deficit/hyperactivity disorder (ADHD) is the most frequently diagnosed neurodevelopmental disorder. The norepinephrine transporter (NET) inhibitor atomoxetine, the first nonstimulant drug licensed for ADHD treatment, also acts as an N-methyl-D-aspartate receptor (NMDAR) antagonist. The compound’s effects on gene expression and protein levels of NET and NMDAR subunits (1, 2A, and 2B) are unknown. Therefore, adolescent Sprague Dawley rats were treated with atomoxetine (3 mg/kg, intraperitoneal injection [ip]) or saline (0.9%, ip) for 21 consecutive days on postnatal days (PND) 21–41. In humans, atomoxetine’s earliest clinical therapeutic effects emerge after 2–3 weeks. Material from prefrontal cortex, striatum (STR), mesencephalon (MES), and hippocampus (HC) was analyzed either directly after treatment (PND 42) or 2 months after termination of treatment (PND 101) to assess the compound’s long-term effects. In rat brains analyzed immediately after treatment, protein analysis exhibited decreased levels of the NET in HC, and NMDAR subunit 2B in both STR and HC; the transcript levels were unaltered. In rat brains probed 2 months after final atomoxetine exposure, messenger RNA analysis also revealed significantly reduced levels of genes coding for NMDAR subunits in MES and STR. NMDAR protein levels were reduced in STR and HC. Furthermore, the levels of two SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, synaptophysin and synaptosomal-associated protein 25, were also significantly altered in both treatment groups. This in vivo study detected atomoxetine’s effects beyond NET inhibition. Taken together, these data reveal that atomoxetine seems to decrease glutamatergic transmission in a brain region-specific manner. Long-term data show that the compound’s impact is not due to an acute pharmacological effect but lasts or even amplifies after a drug-free period of 2 months, leading to altered development of synaptic composition. These alterations might contribute to atomoxetine’s clinical effects in the treatment of ADHD, a neurodevelopmental disorder in which synaptic processes and especially a dysregulated glutamatergic metabolism seem to be involved.

Xenon-induced changes in CNS sensitization to pain.

Electrophysiological investigations of the spinal cord in animals have shown that pain sensitizes the central nervous system via glutamate receptor dependent long-term potentiation (LTP) related to an enhancement of pain perception. To expand these findings, we used functional magnetic resonance (fMRI), blood oxygen level dependent (BOLD) and perfusion imaging in combination with repeated electrical stimulation in humans. Specifically we monitored modulation of somatosensory processing during inhibition of excitatory transmission by ocular application of the glutamate receptor antagonist xenon. BOLD responses upon secondary stimulation increased in mid insular and in primary/secondary sensory cortices under placebo and decreased under xenon treatments. Xenon-induced decreases in regional perfusion were confined to stimulation responsive brain regions and correlated with time courses of xenon concentrations in the cranial blood. Moreover, effects of xenon on behavioral, fMRI and perfusion data scaled with stimulus intensity. The dependence of pain sensitization on sufficient pre-activation reflects a multistage process which is characteristic for glutamate receptor related processes of LTP. This study demonstrates how LTP related processes known from the cellular level can be investigated at the brain systems level.

Diencephalic GABAergic Neurons in vitro Respond to Prolactin with a Rapid Increase in Intracellular Free Calcium

In order to analyze the feedback action of prolactin (PRL) on the hypothalamus on the cellular level, we used primary cultures of rat embryonic diencephalon to measure the calcium response of individual neurons to PRL by means of fast fluorescence photometry. The cultures were subsequently stained with antibodies against the neuronal marker MAP-2, glutamic acid decarboxylase (GAD) or tyrosine hydroxylase (TH). PRL caused a rapid rise of intracellular free Ca2+ in a specific type of GABAergic neuron characterized by a spindle-shaped bipolar morphology and immunoreactivity to MAP-2 and GAD but not to TH. It is concluded that a subpopulation of hypothalamic GABAergic but not dopaminergic neurons react to PRL with a rapid increase in intracellular free Ca2+. These data are compatible with the assumption of a rapid negative feedback regulation of the secretion of PRL from the pituitary mediated by tuberoinfundibular GABAergic neurons.

Formation and characterisation of neuromuscular junctions between hiPSC derived motoneurons and myotubes

Striated skeletal muscle cells from humans represent a valuable source for in vitro studies of the motoric system as well as for pathophysiological investigations in the clinical settings. Myoblasts can readily be grown from human muscle tissue. However, if muscle tissue is unavailable, myogenic cells can be generated from human induced pluripotent stem cells (hiPSCs) preferably without genetic engineering. Our study aimed to optimize the generation of hiPSCs derived myogenic cells by employing selection of CD34 positive cells and followed by distinct, stepwise culture conditions. Following the expansion of CD34 positive single cells under myogenic cell culture conditions, serum deprived myoblast-like cells finally fused and formed multinucleated striated myotubes that expressed a set of key markers for muscle differentiation. In addition, these myotubes contracted upon electrical stimulation, responded to acetylcholine (Ach) and were able to generate action potentials. Finally, we co-cultured motoneurons and myotubes generated from identical hiPSCs cell lines. We could observe the early aggregation of acetylcholine receptors in muscle cells of immature co-cultures. At later stages, we identified and characterised mature neuromuscular junctions (NMJs). In summary, we describe here the successful generation of an iPS cell derived functional cellular system consisting of two distinct communicating cells types. This in vitro co-culture system could therefore contribute to research on diseases in which the motoneurons and the NMJ are predominantly affected, such as in amyotrophic lateral sclerosis or spinal muscular atrophy.

Etomidate reduces glutamate uptake in rat cultured glial cells: involvement of PKA.

Glutamate is the main excitatory neurotransmitter in the vertebrate CNS. Removal of the transmitter from the synaptic cleft by glial and neuronal glutamate transporters (GLTs) has an important function in terminating glutamatergic neurotransmission and neurological disorders. Five distinct excitatory amino‐acid transporters have been characterized, among which the glial transporters excitatory amino‐acid transporter 1 (EAAT1) (glutamate aspartate transporter) and EAAT2 (GLT1) are most important for the removal of extracellular glutamate. The purpose of this study was to describe the effect of the commonly used anaesthetic etomidate on glutamate uptake in cultures of glial cells.