Elektrophysiologische Charakterisierung künstlicher Ionenkanäle in lebenden Zellen

Durch Ausübung physiologischer Grundfunktionen spielen Ionenkanäle eine entscheidende Rolle für die reguläre Funktion von Zellen. Zum besseren Verständnis ihrer Struktur und Funktion sind Untersuchungen natürlicher und künstlicher Ionenkanäle wichtige Werkzeuge. Großes analytisches und therapeutisches Potential ist in der Untersuchung künstlicher Kanäle in lebenden Zellen vorhanden, was bisher wenig Beachtung fand. In der vorliegenden Arbeit wurde die Wirkung der künstlichen Ionenkanäle THF-gram, THF-gram-TBDPS sowie linked-gram-TBDPS auf elektrophysiologische Eigenschaften boviner Trabekelwerkszellen des Auges anhand von Patch-Clamp-Untersuchungen im Whole-Cell-Modus analysiert. Die Untersuchung brachte folgende Erkenntnisse: 1. Die Inkorporation aller drei Verbindungen war erfolgreich, was sich durch Anstieg der Stromdichte und Verschiebung des Umkehrpotentials zeigte. 2. Einbau von THF-gram und THF-gram-TBDPS war mit dem Überleben der Zellen vereinbar, während linked-gram-TBDPS aufgrund einer sehr potenten Antwort bereits bei sehr geringen Konzentrationen zum raschen Zelltod führte. 3. Eine Asymmetrie der Stromantwort zugunsten stärkerer Auswärtsströme wurde bei THF-gram und in schwächerer Ausprägung bei THF-gram-TBDPS festgestellt. Linked-gram-TBDPS zeigte keine derartige Asymmetrie. 4. Unter Verwendung von Cs+ als Ladungsträger war der beobachtete Anstieg der Stromdichte bei allen drei Verbindungen eindeutig stärker als unter physiologischen Bedingungen (Na+/K+). 5. Die dargestellten Erkenntnisse sind ein erster Schritt zur therapeutischen Anwendung von künstlichen Ionenkanälen. Eine Weiterentwicklung in Richtung höherer Selektivität und besserer Kontrolle ist jedoch genauso erforderlich wie die Klärung der klinischen Umsetzbarkeit.Ion channels play a pivotal role for regular cell function. To better understand their structure and function, investigation of both natural and artificial ion channels is being performed to date. Investigation of artificial channels in living cells hides a big potential, however, little attention has been paid to this field so far. In this work, the effect of the artificial ion channels THF-gram, THF-gram-TBDPS and linked-gram-TBDPS on electrophysiological properties of bovine trabecular meshwork cells was investigated with the patch-clamp-technique. Following results were obtained: 1. Incorporation of all three compounds was successful, which was proven by increase of current density and cell depolarisation. 2. The cells survived after incorporation of THF-gram and THF-gram-TBDPS but not after linked-gram-TBDPS, which resulted in cell death at very low concentrations. 3. Larger outward currents were observed with THF-gram and, at a lower extent, with THF-gram-TBDPS. Linked-gram-TBDPS did not show such an asymmetry. 4. With Cs+ as charge carrier all three compunds showed a stronger increase of current density than under physiological conditions (Na+/K+). 5. The decribed results are a first step towards therapeutic application of artificial ion channels, however, further development towards higher selectivity and better control is as necessary as clarification of clinical feasibility

The 4-Aminopyridine Model of Acute Seizures in vitro Elucidates Efficacy of New Antiepileptic Drugs

Up to date, preclinical screening for new antiepileptic substances is performed by a combination of different in vivo models of acute seizures, for which large numbers of animals are necessary. So far, little attention has been paid to in vitro models, which are also able to detect antiepileptic efficacy and in principle could likewise serve for exploratory preclinical screening. One of the established in vitro models of acute seizures is the 4-aminopyridine (4-AP) model. Previous studies have shown that the 4-AP model is capable to recapitulate the antiepileptic efficacy of standard antiepileptic drugs (AEDs) such as valproate or carbamazepine. Here, we employed a dual methodological approach using electrophysiology and optical imaging to systematically test the antiepileptic efficacy of three new-generation AEDs with distinct mechanisms of action (lacosamide, zonisamide, and levetiracetam). We found that frequency of 4-AP induced seizure like events (SLE) was the most sensitive parameter to detect dose-dependent antiepileptic effects in these compounds. Specifically, levetiracetam reduced SLE frequency while lacosamide and zonisamide at higher doses completely blocked SLE incidence. Analysis of the intrinsic optical signal additionally revealed a subiculum-specific reduction of the area involved in the propagation of ictal activity when lacosamide or zonisamide were administered. Taken together, our data adds some evidence that acute seizure models in vitro are in principle capable to detect antiepileptic effects across different mechanisms of action with efficacy similar to acute models in vivo. Further studies with negative controls, e.g., penicillin as a proconvulsant, and other clinically relevant AEDs are needed to determine if this acute in vitro model might be useful as exploratory screening tool. In view of the increasing sensitivity toward animal welfare, an affective in vitro model may help to reduce the number of laboratory animals deployed in burdening in vivo experiments and to preselect substances for subsequent testing in time- and cost-laborious models of chronic epilepsy

Dimethylethanolamine Decreases Epileptiform Activity in Acute Human Hippocampal Slices in vitro

Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy with about 30% of patients developing pharmacoresistance. These patients continue to suffer from seizures despite polytherapy with antiepileptic drugs (AEDs) and have an increased risk for premature death, thus requiring further efforts for the development of new antiepileptic therapies. The molecule dimethylethanolamine (DMEA) has been tested as a potential treatment in various neurological diseases, albeit the functional mechanism of action was never fully understood. In this study, we investigated the effects of DMEA on neuronal activity in single-cell recordings of primary neuronal cultures. DMEA decreased the frequency of spontaneous synaptic events in a concentration-dependent manner with no apparent effect on resting membrane potential (RMP) or action potential (AP) threshold. We further tested whether DMEA can exert antiepileptic effects in human brain tissue ex vivo. We analyzed the effect of DMEA on epileptiform activity in the CA1 region of the resected hippocampus of TLE patients in vitro by recording extracellular field potentials in the pyramidal cell layer. Epileptiform burst activity in resected hippocampal tissue from TLE patients remained stable over several hours and was pharmacologically suppressed by lacosamide, demonstrating the applicability of our platform to test antiepileptic efficacy. Similar to lacosamide, DMEA also suppressed epileptiform activity in the majority of samples, albeit with variable interindividual effects. In conclusion, DMEA might present a new approach for treatment in pharmacoresistant TLE and further studies will be required to identify its exact mechanism of action and the involved molecular targets

Loss of long-term potentiation at hippocampal output synapses in experimental temporal lobe epilepsy

Patients suffering from temporal lobe epilepsy (TLE) show severe problems in hippocampus dependent memory consolidation. Memory consolidation strongly depends on an intact dialog between the hippocampus and neocortical structures. Deficits in hippocampal signal transmission are known to provoke disturbances in memory formation. In the present study, we investigate changes of synaptic plasticity at hippocampal output structures in an experimental animal model of TLE. In pilocarpine-treated rats, we found suppressed long-term potentiation (LTP) in hippocampal and parahippocampal regions such as the subiculum and the entorhinal cortex (EC). Subsequently we focused on the subiculum, serving as the major relay station between the hippocampus proper and downstream structures. In control animals, subicular pyramidal cells express different forms of LTP depending on their intrinsic firing pattern. In line with our extracellular recordings, we could show that LTP could only be induced in a minority of subicular pyramidal neurons. We demonstrate that a well-characterized cAMP-dependent signaling pathway involved in presynaptic forms of LTP is perturbed in pilocarpine-treated animals. Our findings suggest that in TLE, disturbances of synaptic plasticity may influence the information flow between the hippocampus and the neocortex

Lithium inhibits tryptophan catabolism via the inflammation‐induced kynurenine pathway in human microglia

Despite its decades' long therapeutic use in psychiatry, the biological mechanisms underlying lithium's mood-stabilizing effects have remained largely elusive. Here, we investigated the effect of lithium on tryptophan breakdown via the kynurenine pathway using immortalized human microglia cells, primary human microglia isolated from surgical specimens, and microglia-like cells differentiated from human induced pluripotent stem cells. Interferon (IFN)-gamma, but not lipopolysaccharide, was able to activate immortalized human microglia, inducing a robust increase in indoleamine-2,3-dioxygenase (IDO1) mRNA transcription, IDO1 protein expression, and activity. Further, chromatin immunoprecipitation verified enriched binding of both STAT1 and STAT3 to the IDO1 promoter. Lithium counteracted these effects, increasing inhibitory GSK3 beta(S9) phosphorylation and reducing STAT1(S727) and STAT3(Y705) phosphorylation levels in IFN-gamma treated cells. Studies in primary human microglia and hiPSC-derived microglia confirmed the anti-inflammatory effects of lithium, highlighting that IDO activity is reduced by GSK3 inhibitor SB-216763 and STAT inhibitor nifuroxazide via downregulation of P-STAT1(S727) and P-STAT3(Y705). Primary human microglia differed from immortalized human microglia and hiPSC derived microglia-like cells in their strong sensitivity to LPS, resulting in robust upregulation of IDO1 and anti-inflammatory cytokine IL-10. While lithium again decreased IDO1 activity in primary cells, it further increased release of IL-10 in response to LPS. Taken together, our study demonstrates that lithium inhibits the inflammatory kynurenine pathway in the microglia compartment of the human brain

In vitro and in vivo anti-epileptic efficacy of eslicarbazepine acetate in a mouse model of KCNQ2-related self-limited epilepsy

Background and purpose: The KCNQ2 gene encodes for the Kv 7.2 subunit of non-inactivating potassium channels. KCNQ2-related diseases range from autosomal dominant neonatal self-limited epilepsy, often caused by KCNQ2 haploinsufficiency, to severe encephalopathies caused by KCNQ2 missense variants. In vivo and in vitro effects of the sodium channel blocker eslicarbazepine acetate (ESL) and eslicarbazepine metabolite (S-Lic) in a mouse model of self-limited neonatal epilepsy as a first attempt to assess the utility of ESL in the KCNQ2 disease spectrum was investigated. Experimental approach: Effects of S-Lic on in vitro physiological and pathological hippocampal neuronal activity in slices from mice carrying a heterozygous deletion of Kcnq2 (Kcnq2+/- ) and Kcnq2+/+ mice were investigated. ESL in vivo efficacy was investigated in the 6-Hz psychomotor seizure model in both Kcnq2+/- and Kcnq2+/+ mice. Key results: S-Lic increased the amplitude and decreased the incidence of physiological sharp wave-ripples in a concentration-dependent manner and slightly decreased gamma oscillations frequency. 4-Aminopyridine-evoked seizure-like events were blocked at high S-Lic concentrations and substantially reduced in incidence at lower concentrations. These results were not different in Kcnq2+/+ and Kcnq2+/- mice, although the EC50 estimation implicated higher efficacy in Kcnq2+/- animals. In vivo, Kcnq2+/- mice had a lower seizure threshold than Kcnq2+/+ mice. In both genotypes, ESL dose-dependently displayed protection against seizures. Conclusions and implications: S-Lic slightly modulates hippocampal oscillations and blocks epileptic activity in vitro and in vivo. Our results suggest that the increased excitability in Kcnq2+/- mice is effectively targeted by S-Lic high concentrations, presumably by blocking diverse sodium channel subtypes

Human gestational N‐methyl‐d‐aspartate receptor autoantibodies impair neonatal murine brain function

Objective: Maternal autoantibodies are a risk factor for impaired brain development in offspring. Antibodies (ABs) against the NR1 (GluN1) subunit of the N-methyl-d-aspartate receptor (NMDAR) are among the most frequently diagnosed anti-neuronal surface ABs, yet little is known about effects on fetal development during pregnancy. Methods: We established a murine model of in utero exposure to human recombinant NR1 and isotype-matched nonreactive control ABs. Pregnant C57BL/6J mice were intraperitoneally injected on embryonic days 13 and 17 each with 240μg of human monoclonal ABs. Offspring were investigated for acute and chronic effects on NMDAR function, brain development, and behavior. Results: Transferred NR1 ABs enriched in the fetus and bound to synaptic structures in the fetal brain. Density of NMDAR was considerably reduced (up to -49.2%) and electrophysiological properties were altered, reflected by decreased amplitudes of spontaneous excitatory postsynaptic currents in young neonates (-34.4%). NR1 AB-treated animals displayed increased early postnatal mortality (+27.2%), impaired neurodevelopmental reflexes, altered blood pH, and reduced bodyweight. During adolescence and adulthood, animals showed hyperactivity (+27.8% median activity over 14 days), lower anxiety, and impaired sensorimotor gating. NR1 ABs caused long-lasting neuropathological effects also in aged mice (10 months), such as reduced volumes of cerebellum, midbrain, and brainstem. Interpretation: The data collectively support a model in which asymptomatic mothers can harbor low-level pathogenic human NR1 ABs that are diaplacentally transferred, causing neurotoxic effects on neonatal development. Thus, AB-mediated network changes may represent a potentially treatable neurodevelopmental congenital brain disorder contributing to lifelong neuropsychiatric morbidity in affected children

Synaptic and intrinsic mechanisms of neuronal excitability

Störungen neuronaler Erregbarkeit sind an vielen neurologischen und psychiatrischen Erkrankungen beteiligt, folglich basieren eine Reihe pharmakologischer Ansätze zur Therapie dieser Erkrankungen auf der Beeinflussung dieser Erregbarkeit. In der vorliegenden Arbeit sind Mechanismen der Erregbarkeit auf synaptischer und intrinsischer Ebene näher untersucht worden. Bezüglich synaptischer Erregbarkeit wurde gezeigt, dass die im Subikulum exprimierten Pyramidenzelltypen, „Burst“- und „Regular“-Zellen, an den CA1-Subikulum Synapsen neben der zellspezifischen Langzeitpotenzierung (LTP) auch eine niedrigfrequenzinduzierte, zellspezifische Langzeitdepression (LTD) zeigen, und dass diese über muskarinerge Acetylcholinrezeptoren, spannungsabhängige Kalziumkanäle und sowohl metabotrope als auch ionotrope Glutamatrezeptoren reguliert wird. Zusätzlich konnte erstmalig gezeigt werden, dass auch Synapsen zwischen Entorhinalkortex (EC) und Subikulum LTP und LTD exprimieren, dabei wurde auch eine neue Form der heterosynaptischen Plastizität zwischen EC-Subikulum und CA1-Subikulum Synapsen beschrieben, welche auf GABAerger LTD beruht. Die beschriebenen Mechanismen sind für das Verständnis synaptischer Erregbarkeit an der hippokampalen Ausgangsstruktur essentiell. Die Regulation der intrinsischen Erregbarkeit wurde an zwei Beispielen untersucht, dem Kv7.5 Kaliumkanal und dem Anoctamin2 Chloridkanal. Wir konnten erstmalig nachweisen, dass Kv7.5 im Gegensatz zu Kv7.2 und Kv7.3 vorwiegend die Erregbarkeit hemmender Neurone kontrolliert, und dass beim Funktionsverlust von Kv7.5 die GABAerge Inhibition erhöht ist. Da unspezifische Kv7 Kanalaktivatoren als Antikonvulsiva eingesetzt werden, haben die hier gewonnenen Erkenntnisse eine direkte klinische Relevanz. Schließlich konnten wir mit Anoctamin2-Chloridkanal die molekulare Identität des kalziumaktivierten Chloridkanals in sensorischen olfaktorischen Neuronen aufklären und zeigen, dass entgegen der bis dahin gängigen Lehrmeinung der kalziumaktivierte Chloridkanal für die adäquate Funktion des Geruchsinns nicht zwingend notwendig ist. Zusammenfassend ergänzen die Arbeiten durch Charakterisierung bis dahin unbekannter zellspezifischer, eingangspezifischer und heterosynaptischer Effekte die bisherige Kenntnis über Mechanismen synaptischer Erregbarkeit und Plastizität im Subikulum. Ferner wird anhand der Funktionsanalyse von Kv7.5 und Anoctamin2 deutlich, dass Steigerung intrinsischer Erregbarkeit abhängig vom Zelltyp entgegengesetzte Effekte auf die Gesamterregbarkeit eines Netzwerks haben kann (Kv7.5) und dass selbst eine Abschwächung der intrinsischen Erregbarkeit nicht zwingend mit einem physiologischen Funktionsverlust einhergehen muss (Anoctamin2).Disturbances of neuronal excitability can lead to a variety of neurological diseases. In the current work, specific aspects of synaptic and intrinsic mechanisms of neuronal excitability were investigated. Regarding synaptic mechanisms, novel cell-specific, input-specific and heterosynaptic mechanisms of synaptic plasticity in the subiculum were characterized. The described mechanisms complement the knowledge about synaptic plasticity at the hippocampal output. With respect to intrinsic excitability, the functional analysis of the Kv7.5 potassium channel demonstrated that depending on the cell type, increase of intrinsic excitability can have opposite effects on network excitability. Finally, the characterization of the Anoctamin2 calcium dependent chloride channel in olfactory neurons showed that, in contrast to previous assumptions, decrease of excitability is not necessarily associated with loss of physiological function and that Anoctamin2 is not required for olfaction

Ca2+-activated Cl- currents are dispensable for olfaction

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