Anti-epileptic and neuroprotective mechanisms by Topiramate and Levetiracetam evaluated in primary cultures

Abstract

The aim of this thesis was to evaluate plausible intracellular mechanisms of action for antiepileptic drugs, Topiramate (TPM) and Levetiracetam (LEV), and to investigate if TPM possesses neuroprotective properties. The thesis is based on results from calcium imaging, immunohistochemical stainings, viability tests, and Western blot using primary cortical or hippocampal cultures, or hippocampal brain slices. The results demonstrate that TPM protects neurons from glutamate (Glu) and kainate induced excitotoxicity in mixed neuronal/astroglial cortical cultures. Further it was shown that this neuroprotective effect could be in part due to TPMs ability to inhibit the accumulation of free Ca2+ and that this ability is inversely related to the level of protein kinase A (PKA) mediated phosphorylation of kainate activated receptors. This thesis also demonstrates that TPM inhibits AMPA induced accumulation of free Ca2+ in astrocytes and that TPM inhibits PKA mediated phosphorylation of the GluR1 subunit of AMPA receptors in astrocytes. The novel antiepileptic drug LEV has been reported to exert several non-conventional effects on neurons and special attention has been directed whether LEV opposes the release of Ca2+ from intraneuronal stores. The results demonstrate that LEV was able to prevent caffeine induced ryanodine receptor mediated Ca2+ release from intracellular Ca2+ stores from hippocampal cultures. Cells treated with the R-enantiomer of LEV (ucb LO60) had no effect on caffeine induced intracellular calcium transients ([Ca2+]i) transients. Levetiracetam significantly inhibited the amplitudes and the number of caffeine induced repeated population spikes and delayed the appearance of spontaneous bursts in rat hippocampal slices in vitro while ucb LO60 was devoid of anticonvulsant activity. The results presented in this thesis demonstrate that TPM protects neurons against Glu and kainate induced excitotoxicity and provide evidence that TPM modulates phosphorylation of kainate activated receptors in both neurons and astrocytes. This thesis also points to that the inhibition of caffeine induced Ca2+ release from intra-neuronal stores might be an excitability-reducing effect of LEV, contributing to its antiepileptic activity.