INVESTIGATION OF THE PATHOPHYSIOLOGY OF MIGRAINE USING FAMILIAL HEMIPLEGIC MIGRAINE MOUSE MODELS

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Metadata	Details
Publication Date	2015-02-01
Journal	Padua@research (University of Padova)
Authors	Clizia Capuani

Abstract

Migraine is a common, highly disabling episodic neurological disorder affecting more than 10% of the population and arises from a primary brain dysfunction that leads to episodic activation and sensitization of the trigeminovascular pain pathway. \nFamilial hemiplegic migraine (FHM) is a rare and very severe monogenic autosomal dominant subtype of migraine with aura. Apart from the motor aura and the possible longer duration of the aura, typical FHM attacks resemble migraine with aura attacks (Pietrobon and Moskowitz, 2013); thus, FHM can be considered as a model of the common forms of migraine. Gain-of-function missense mutations in CACNA1A gene, encoding the pore-forming subunit of the neuronal voltage-gated Ca2+ channel CaV2.1 (also known as P/Q-type Ca2+ channel), cause FHM type 1 (FHM1) and loss-of-function mutations in ATP1A2, the gene encoding the astrocytic Na,K-ATPase Î±2 subunit, cause FHM type 2 (FHM2) (Ophoff et al., 1996; De Fusco et al., 2003). FHM knock-in (KI) mice carrying mutations causing either FHM1 or FHM2 show facilitation of induction and propagation of experimental CSD (van den Maagdenberg et al., 2004, 2010; Leo et al., 2011), the phenomenon underlying migraine aura and a key triggering event for trigeminovascular activation.\nFHM1 KI mice, carrying the R192Q mutation, show an increased influx of Ca2+ through P/Q-type Ca2+ channels in neurons, including cortical pyramidal cells, and an increased probability of glutamate release at cortical pyramidal cells synapses (Pietrobon, 2010; Tottene et al., 2009), that may explain the facilitation of experimental CSD. Recent findings in our laboratory (Fabbro, Sessolo, Vecchia and Pietrobon, manuscript in preparation) show that the frequency of the up-states recorded in acute cortical slices, that resemble slow oscillation in vivo (Steriade et al., 1993), is larger in FHM1 KI than WT mice, suggesting that the gain-of-function of P/Q-type Ca2+ channels facilitates the mechanisms of up-states generation and/or decreases the refractory period after an up-state.\nThe first aim of my work was to further investigate the role of P/Q-type Ca2+ channels in the recurrent network activity underlying the up-states in WT mice. I studied the effect of pharmacological inhibition of P/Q-type Ca2+ channels on the up-state activity recorded from layer 2/3 pyramidal neurons in acute slices of mouse somatosensory cortex, by performing single and double patch clamp experiments. I found that the block of P/Q-type Ca2+ channels transforms the up-states into events resembling interictal epileptiform discharges. I evaluated the mean excitatory and inhibitory synaptic conductances (Ge and Gi) during the control up-states, during the simil-interictal epileptiform events after P/Q-type Ca2+ channels block as well as immediately after the application of the P/Q-type Ca2+ channels blocker when the channels were not completely blocked. I showed that 1) P/Q-type Ca2+ channels have a dominant role in controlling both excitatory and inhibitory synaptic transmission onto pyramidal cells during the spontaneous recurrent network activity that underlies the up-states. However, the block of P/Q-type Ca2+ channels reduces recurrent inhibition more than recurrent excitation and shifts the cortical excitation-inhibition balance towards excitation. 2) When, as a consequence of the block of P/Q-type Ca2+ channels, Ge/Gi increases above a critical value, the spontaneous network activity changes and the up-states are transformed into events resembling simil-interictal epileptiform discharges. These data suggest that, in the cerebral cortex, P/Q-type Ca2+ channels play a more prominent role in controlling inhibitory compared to excitatory synaptic transmission.\nGiven that at many cortical synapses P/Q- and N-type (also known as CaV2.2) Ca2+ channels cooperate in controlling synaptic transmission, I also investigated the effect of blocking N-type Ca2+ channels on up-state activity. Pharmacological inhibition of this channel strongly reduces the up-states frequency, indicating a role for N-type Ca2+ channel in controlling up-states frequency. After the block of N-type Ca2+ channels, Ge/Gi increases but not sufficiently to transform up-states in simil-interictal epileptiform events.\nThe aim of my second project was to investigate the unknown mechanisms underlying facilitation of experimental CSD in FHM2 KI mice. After setting the conditions in which facilitation of CSD was observed in vitro, I studied three possible mechanisms that may underlie the facilitation of CSD in heterozygous FHM2 KI mice, in acute slices of mouse somatosensory cortex. \nGiven the specific localization and functional coupling of the Î±2 Na,K-ATPase to glutamate transporters in astrocyte processes surrounding cortical glutamatergic synapses (Cholet et al., 2002), I first investigated whether the loss-of-function of Î±2 Na,K-ATPase results in an impaired astrocyte-mediated clearance of glutamate from the synaptic cleft during cortical neuronal activity. I monitored the rate of glutamate clearance electrophysiologically, by measuring the synaptically-activated glutamate transporter-mediated current (STC) evoked in astrocytes of layer 1 by extracellular stimulation of neuronal afferents in the same layer. Either single pulse stimulation or trains of stimuli at high frequencies (50 and 100 Hz) were delivered. I isolated the STC pharmacologically in order to measure the STC decay time course that provides a relative measure of the glutamate clearance by astrocytes (Bergles and Jahr, 1997; Diamond and Jahr, 2000). I found that the clearance of glutamate release is slower in FHM2 KI compared to WT mice. The slowing of glutamate clearance was more pronounced after a train stimulation than a single stimulus and increased with increasing frequency of the train. My data show that the loss-of-function of Î±2 Na,K-ATPase results in an impairment of glutamate clearance and suggest that the impairment increases with increasing frequency of cortical activity. \nSurprisingly, the STC amplitude after a single pulse stimulation was higher in FHM2 KI than in WT mice. Given that the STC amplitude is proportional to the glutamate release evoked at the synapses by the extracellular stimulation (Bergles and Jahr, 1997; Diamond and Jahr, 2000), this result would suggest that the extracellular stimulation elicits a larger glutamate release in FHM2 KI than in WT mice. Indeed, during repetitive stimulation the STC depressed more in FHM2 KI than WT mice, as expected if the probability of glutamate release is increased in the mutant mice. \nGiven the key role of NMDA receptors in the positive feedback cycle, that ignites CSD (Tottene et al., 2011; Pietrobon and Moskowitz, 2014), both the reduced clearance of glutamate and the increased glutamate release may be implicated in the facilitation of CSD in FHM2 KI mice.\nGiven that most models of CSD include local increase of extracellular K+ concentration above a critical value, as a triggering event in the initiation of CSD (Pietrobon and Moskowitz, 2014), and that pharmacological evidence indicates that Î±2 and/or Î±3 Na,K-ATPase participate in the clearance of K+ from the extracellular space during intense neuronal activity (DâAmbrosio et al., 2002; Kofuji and Newman, 2004), I investigated whether K+ clearance by astrocytes is impaired in FHM2 KI mice. I evaluated the rate of K+ clearance from the interstitial space, by recording the slowly decaying current, which is mainly due to K+ influx through Kir channels, evoked in layer 1 astrocytes by extracellular stimulation. I measured the decay time course of this current, which provides an indirect measure of the K+ clearance rate by astrocytes. Preliminary experiments show that the decay time course of the K+ current evoked by train stimulation is similar in WT and FHM2 KI mice. If confirmed, this result would indicate that there are no changes in the rate of K+ clearance in FHM2 KI mice compared to WT.\nGiven that Î±2 Na,K-ATPase is tightly coupled to the Na+/Ca2+ exchanger at plasma membrane microdomains that overlay the endoplasmic reticulum (Lencesova et al., 2004; Golovina et al., 2003) and hence its loss-of-function could influence Ca2+ homeostasis, we investigated whether the Ca2+ content in the intracellular Ca2+ stores of astrocytes in FHM2 KI mice is increased. We obtained an indirect measure of the amount of Ca2+ in the stores, by measuring in cultured cortical astrocytes the Ca2+ transient induced by ionomycin in Ca2+-free medium. This transient was larger in FHM2 KI mice compared to WT mice, indicating that the Ca2+ content is increased in the intracellular Ca2+ stores of astrocytes in FHM2 KI mice. \nI measured CSD threshold and velocity after depletion of intracellular Ca2+ stores by CPA, a SERCA inhibitor, and I observed that depletion of Ca2+ stores reduces the facilitation of CSD in FHM2 KI mice, without affecting CSD in WT mice. This result suggests a role of increased Ca2+ concentration within the astrocytes intracellular stores in the facilitation of experimental CSD in FHM2 KI mice. \n

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