Elektrophysiologische Charakterisierung neuronaler spannungsabhängiger Calciumströme des Locus coeruleus in zwei experimentellen Parkinson-Modellen der Maus

Der Locus coeruleus (LC) ist ein noradrenerger Kern des dorsolateralen Hirnstammes, der auf vielfältige Weise in die physiologischen Prozesse des zentralen Nervensystems eingebunden ist. Eine funktionelle Beeinträchtigung dieses Kerngebietes lässt sich bei vielen neurologischen und psychiatrischen E...

Full description

Saved in:
Bibliographic Details
Main Author: Griesbach, Markus
Contributors: Decher, Niels (Prof. Dr. phil. nat.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2023
Online Access:PDF Full Text
Tags: Add Tag
No Tags, Be the first to tag this record!

The Locus coeruleus (LC) is a noradrenergic nucleus of the dorsolateral brainstem, that is an integral part of the electrophysiological processes of the central nervous system. Functional impairment of this region can be detected in various neurological and psychiatric disorders, oftentimes even in early stages, so that the LC is of great diagnostic and therapeutic interest. In the pathogenesis of Parkinson’s disease (PD), dopaminergic neurons of the Substantia nigra pars compacta (SNpc) gradually degenerate, leading to the characteristic motor symptoms of this chronic neurological disorder. Neurons of the LC are affected by neurodegeneration even before the SNpc. The progredient loss of the noradrenergic LC-neurons is discussed as one of the main reasons for the non-motor symptoms of PD. Furthermore, neurons of the LC seem to modify the electrophysiological properties of the SNpc in a way that is actually neuroprotective. Causes for the progredient degeneration and selective vulnerability of the LC are mostly unknown though. In order to ensure a steady release of noradrenalin (NA), LC-neurons are characterized by a distinctice periodicity of their depolarisations. The underlying pacemaking mechanisms of the LC are primarily relying on voltage-dependent calcium channels. Previous studies have shown that neurons of the LC show a higher frequency of depolarisations within the course of PD. The resulting rise in intracellular calcium concentration, as well as oxidative stress and the characteristic alpha-synuclein aggregates in the cytosol of the affected cells, have been suggested as possible factors of the pathogenesis of PD. Stabilizing the properties of cellular calcium-homeostasis might therefore have positive effects on the progression of PD. This thesis investigated the influence of two different experimental models of PD on the electrophysiological properties of voltage-dependent calciumcurrents of the LC. The first experimental model evaluated brain slices of wildtype-mice (C57/Bl6), that were previously incubated in a solution of rotenone for a predefined time period. Rotenone, a substance best known for its use as an insecticide, inhibits complex I of the mitochondrial chain of oxidative phosphorylation, hence causing a situation of oxidative and metabolic cellular stress as it is also observed in cells that are affected by PD. In order to isolate the calcium currents of the inspected cells, Tetrodotoxin (TTX), Tetraethylammonium (TEA) and 4-Aminopyridine (4-AP) were utilized as pharmacological antagonists of voltage-dependent sodium- and potassium channels. The calcium-currents of these neurons were then recorded, utilizing the patch-clamp method in whole-cell configuration. The results of these recordings have shown a significant reduction in amplitude of the inward calcium-currents in the rotenone-group. The samples of the rotenone-group were then perfused with a highly concentrated cobalt(II)-chloride-solution in order to antagonize the remaining calcium-currents. Although the inward calcium-currents could be significantly reduced by this procedure, they could not be totally blocked, suggesting the existence of a subset of calcium-channels unsensitive to cobalt-specific antagonization. The remaining component of the current therefore seems to be unsensitive to cobaltspecific antagonization, suggesting that either another subset of voltage-dependent calcium-channels or for example TRP-channels might play an additional role in that regard. The second experimental model evaluated the electrophysiological influence of cytosolic alpha-synuclein-aggregates as they typically appear in LC-neurons of patients with PD, with a focus on possible variations of voltage-dependent calcium-currents. To accomplish this, three randomized groups of wildtype-mice (C57/Bl6) were unilaterally injected with a viral vector system in the dorsolateral pons-region utilizing a stereotactic application system. Depending on the group, the viral vector system contained either the gene of a wildtype-alpha-synuclein, the gene of the mutant type alpha-synuclein A53T or the gene of the enzyme Luciferase. A53T is a variant of alpha-Synuclein that shows a significantly higher rate of aggregation than wildtype-alpha-synuclein. The Luciferase group was utilized as the control group for this experiment. After a predefined time period of four months, single-cell recordings of LC-neurons were conducted with the same method that was used in the aforementioned first model system. To again isolate the inward calcium-currents of the inspected neurons, Tetrodotoxin (TTX), Tetraethylammonium (TEA) and 4-Aminopyridine (4-AP) were utilized as pharmacological antagonists of voltage-dependent sodium- and potassium channels. Comparable to the results of the first, rotenone-based model system, a significant decrease of inward calcium-currents could be shown in the wildtype-alpha-synuclein group as well as the A53T-alpha-synuclein group. The reduced calciuminflux might in both model systems be caused by an elevated intracellular calcium-load, possibly caused by a combination of increased release of calcium from intracellular storages as well as an increased frequency of calcium-dependent depolarisations of these LC-neurons. To further investigate the origin of the increased intracellular calcium-load, calcium-imaging experiments might be conducted as a follow-up to this thesis. These results suggest, that a pharmacological modulation of voltage-dependent calciumchannels of LC-neurons might be beneficial to patients with early stages of PD.