Investigation of the subcortical spreading mechanisms of the optogenetically induced cortical spreading depression in mice

Uzay B. Investigation of the Subcortical Spreading Mechanisms of the Optogenetically Induced Cortical Spreading Depression in Mice, Hacettepe University Graduate School of Health Sciences Basic Neurological Sciences (Neuroscience) Program, Doctor of Philosophy Thesis, Ankara, 2019. Migraine is a neurological disorder characterized by severe headache attacks. Cortical spreading depression (CSD) is known to be the electrophysiological correlate of migraine aura. CSD results in opening of pannexin-1 megachannels that release ATP in the cortex and triggers parenchymal neurogenic inflammation that precedes meningeal inflammation which is thought to cause migraine headache. Symptoms suggesting subcortical dysfunction are observed during migraine attacks and subcortical structures are known to be affected. It is known that CSD spreads to the subcortical structures, whose mechanisms are yet not completely elucidated. In our study, the effects of purinergic P2X7 receptors on the subcortical spread of CSD and the response of subcortical structures following CSD were investigated using a potent and selective P2X7R antagonist. Experiments were performed in male C57BL6 transgenic mice of the Thy1-ChR2 genotype by inducing CSD optogenetically. Optogenetic CSD threshold was 17.9 mJ, values were accumulated below 30 mJ threshold, and there was no difference between heterozygous and homozygous mice. P2X7R antagonism had no effect on the CSD threshold. In the hypothalamic electrophysiological recordings that were done while triggering CSD, a direct current shift was observed at a mean amplitude of 0.8 mV with a mean delay of 51 seconds. P2X7R antagonism had no effect on this direct current shift. In subcortical structures that were investigated (thalamus, hypothalamus, striatum, hippocampus) and in cortex, expression of P2X7R receptor was increased after CSD, NF-kappa B-p65 was translocated to the nucleus in the astrocytes and c-fos positivity was increased in the hypothalamus. These effects could all be reversed by P2X7R antagonism. In order to investigate neurogenic inflammation in the cortex following CSD in an in vivo setup, an adeno-associated virus (AAV) carrying 'NF-κB-RE-EGFP' construct was injected intracortically, CSD was induced by using 1 M KCl-soaked cotton balls and the cortex was imaged by confocal microscope. The preliminary results showed an increase in the signal that was not observed under P2X7R antagonism. In conclusion, P2X7 receptors play an important role in neuronal activation in the hypothalamus after CSD, in the neurogenic inflammation in the cortex and subcortical structures; however, it may not have a direct role in the electrophysiological spread of CSD to subcortical structures. P2X7 receptors may be used as a target in the prophylaxis and treatment of migraine.