Die Cav- und KCa/SK- Ionenkanalfamilien in Locus Coeruleus Neuronen der Maus - Funktionelle Charakterisierung und Implikationen für die Parkinson-Erkrankung

Der Locus Coeruleus (LC) ist ein noradrenerger Kern des Hirnstammes, der an der Regulation vielfältiger, physiologischer Prozesse beteiligt ist. Störungen des LC-Noradrenalin Systems sind in der Pathogenese verschiedener psychiatrischer und neurodegenerativer Erkrankungen beteiligt und sind ein früh...

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Bibliographic Details
Main Author: Matschke, Lina
Contributors: Decher, Niels (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2016
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The Locus Coeruleus (LC) is a noradrenergic nucleus of the brainstem that plays a major role in the regulation of versatile physiological processes. Dysfunction of the LC noradrenergic system is involved in psychiatric and neurodegenerative diseases and is an early hallmark of Parkinson´s disease (PD). While degeneration of dopaminergic Substantia Nigra pars compacta (SNpc) neurons accounts for the motor symptoms observed in PD patients, the extensive loss of noradrenergic LC neurons is responsible for most of the non-motor symptoms that occur in early stages of the disease. However, the reasons why LC neurons are selectively vurnerable during the pathogenesis of PD are only poorly understood. To warrant a permanent release of Noradrenaline LC neurons possess an intrinsic pacemaking mechanism, which is ultimately coupled to cell survival signaling pathways. It is suggested that activity-dependent Ca2+ Influx, mediated by L-type Ca2+ channels, leads to mitochondrial oxidant stress in LC neurons and other PD-related brain regions. In addition, a neuroprotective function of Ca2+ activated potassium channels that modulate pacemaking of dopaminergic SNpc neurons, is proposed. Therefore, the analysis of ion channels underlying the autonomous electrical activity of LC neurons can lead to a better understanding of the vulnerability of these neurons. In the present study, I performed RT-PCR expression analysis and utilized patch-clamp recordings of in vitro brainstem slices to characterize the molecular composition and function of distinct ion channel families in mouse LC neurons. First, a profile regarding the electrophysiological characteristics and the expression of potassium selective ion channels in LC neurons was compiled. These analyses revealed an electrophysiological phenotype of LC neurons that was marked by regular, broad action potentials with pronounced afterhyperpolarizations fluctuating around a depolarized membrane potential. Utilizing RT-PCR expression analyses the molecular composition of voltage dependent potassium channels that most probably mediate the A-type K+ and the persistent K+ currents of LC neurons was elucidated. Among others, the A-type channels Kv4.3 and Kv1 in combination with Kvβ1 were detected. These channels are modulated by the oxidative potential of a cell and could therefore play a role during pathological conditions where mitochondrial function is impaired. In addition, expression of the GIRK channel subunits GIRK-1 and GIRK-4 as well as distinct K2P channel subunits, that are involved in setting the resting membrane potential of central neurons, was detected. In the course of the functional characterization of voltage dependent Ca2+ channels I utilized RT-PCR expression analyses as well as slice patch clamp recordings in combination with L-type and T-type Ca2+ channel blockers. These experiments showed the expression of both Cav1 and Cav3 subtypes in LC neurons mediating a pronounced low-voltage activated Ca2+ conductance. Analyzing action potential trains, I revealed that neither L-type nor T-type Ca2+ channel antagonism alone leads to a change in firing frequency or action potential properties. However, a combined application of antagonists significantly decreased the afterhyperpolarization, resulting in an increased firing frequency. Hence, I report for the first time the functional expression of T-type Ca2+ channels in LC neurons and demonstrate their role in modulating the pacemaking mechanism of LC neurons by working in concert with L-type Ca2+ channels. Next to L-type Ca2+ channels, T-type Ca2+ channels should therefore be taken into account as potential candidates in mediating activity-dependent oxidant stress under pathological conditions. In the course of the functional characterization of Ca2+ activated potassium channels in LC neurons the expression of the SK channel subtypes SK1, SK2 and SK3 was revealed. Using slice patch clamp recordings in combination with the selective SK channel antagonist Apamin and the positve SK channel modulator NS309 I display that SK channels mediate K+ outward currents that flow during the afterhyperpolarization of LC neurons. Recordings of spike trains elucidated that inhibition of SK channels leads to decreased afterhyperpolarizations and an increased firing frequency whereas activation of these channels results in augmented afterhyperpolarizations and a decelerated firing frequency. Hence, SK channels can be considered as important regulators of LC neuron pacemaking. Using Calcium Imaging experiments in in vitro models of glutamate and rotenone toxicity I revealed that the pharmacological SK channel activation prevents a dysregulation of the intracellular Ca2+ homeostasis. Additionally, my patch clamp experiments demonstrated for the first time that acute rotenone exposure induces a significant depolarisation and an increase of firing frequency in LC neurons which could be prevented by SK channel activation. Eventually, stereological analyses showed that SK channel activation via NS309 significantly counteracts the degeneration of LC neurons induced by toxic concentrations of rotenone in vitro. Thus, the activation of SK channels is proposed as a promising target to protect LC neurons in early stages of the PD pathology.