Mechanisms of susceptibility to cortical spreading depression in migraine

Migraine is a common and disabling brain disorder, characterized by recurrent attacks of unilateral and throbbing headache, that in 30% of patients is preceded by transient sensory disturbances, the migraine aura. The neurophysiological correlate of migraine aura is the cortical spreading depression (CSD), a slow and self-sustaining propagating wave of complete depolarization of brain cells. A key unanswered question in migraine neurobiology concerns the mechanisms that make the brain of migraineurs susceptible to CSD. Important insights into these mechanisms can be obtained by studying the molecular and cellular mechanisms of CSD initiation experimentally induced in normally metabolizing brain slices, and the mechanisms underlying the facilitation of CSD initiation in genetic mouse models of the disease. I investigated these mechanisms in WT and in knock-in mice carrying a gain-of-function mutation in the CaV2.1 calcium channel, which causes familial hemiplegic migraine type 1 (FHM1, a rare form of migraine with aura), and leads to enhanced glutamate release at cortical synapses and to a lower stimulation threshold for CSD induction. High KCl puffs of increasing duration up to the threshold duration eliciting a CSD were applied on layer 2/3 of cerebral cortex slices whilst the membrane potential of a pyramidal neuron located near the CSD initiation site (100μm) and the intrinsic optic signal were simultaneously recorded before and after application of a specific inhibitor of different ion channels (NMDARs, CaV channels, NaV channels and the persistent Na+ current). The most important results obtained are the following. i) The mechanism underlying the ignition of CSD by a threshold stimulus and not by a just subthreshold stimulus is the CaV-dependent activation of a threshold level of NMDARs. ii) The delay of several seconds with which this occurs underlies the delay of CSD initiation relative to the rapid neuronal depolarization produced by the KCl stimulus. iii) The threshold level of NMDAR activation that ignites CSD is quantitatively similar in FHM1 and WT mice, but is reached with a stimulus of much lower intensity and in a shorter time in FHM1 mice, thus explaining the facilitation of CSD initiation in the mutants. iv) No matter the intensity of the stimulation, CaV channels are necessary for CSD initiation in both WT and FHM1 mice. v) While in WT mice the inhibition of NMDARs prevents CSD initiation by threshold and even largely suprathreshold stimuli, in FHM1 mice largely suprathreshold stimuli can trigger a CSD, suggesting that, besides activation of NMDARs, other CaV2.1-dependent processes may contribute to CSD initiation by intense stimulation in FHM1 mice. vi) The NaV channels responsible for the persistent Na+ current play a role in the CSD initiation (not in propagation) but are not necessary, because increasing the depolarizing stimulus the CSD ignites. In this PhD project I also studied the effect of sleep deprivation, a common migraine trigger, on CSD initiation in WT and knock-in mice, carrying a loss-of-function mutation in the astrocytic α2 Na+/K+ ATPase which causes familial hemiplegic migraine type 2 (FHM2). This mutation leads to reduced rate of K+ and glutamate clearance at cortical synapses and a lower stimulation threshold for CSD induction. The mice were sleep deprived for 6 hours with the gentle handling method, to minimize the stress. The most important results obtained are the following. i) Sleep deprivation has no effect on CSD threshold induction and velocity of propagation in WT mice. ii) Sleep deprivation decreases the threshold for CSD induction and increases the velocity of CSD propagation in FHM2 mice. iii) The sleep deprivation further enhances the CSD facilitation in FHM2 compared to WT mice.