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Familial Hemiplegic Migraine type 2 (FHM2) is a rare subtype of migraine with aura, caused by loss-of-function mutations in ATP1A2, the gene encoding for the alpha2 subunit of the Na+,K+-ATPase (NKA), which is expressed almost exclusively in astrocytes in the adult brain. In the brain of heterozygous knockin (KI) mice carrying the W887R FHM2 mutation, the alpha2 NKA protein is halved compared to WT. These mice show a lower threshold for induction and a higher velocity of propagation of experimental cortical spreading depression (CSD), the phenomenon underlying migraine aura. The aim of my PhD project was to investigate the unknown mechanisms leading to CSD facilitation in FHM2 mouse model. Given the colocalization of alpha2 NKA and the glial glutamate transporters in the astrocytic processes surrounding glutamatergic synapses, the working hypothesis is that glutamate clearance is impaired in FHM2-KI mice, thus leading to enhanced glutamatergic neurotransmission and hence CSD facilitation. By measuring the synaptically activated glutamate transporter current in cortical astrocytes, the lab demonstrated that the reduced expression of the alpha2 NKA in FHM2-KI mice leads to a reduced rate of glutamate clearance. We demonstrate that, in vitro, experimental CSD is facilitated in FHM2-KI compared to WT mice. I therefore investigated whether there is a causative link between the impaired glutamate clearance and CSD facilitation in FHM2-KI mice, using two different approaches: first, testing whether Ceftriaxone (Cef), a drug that increases the membrane expression of GLT-1 in neocortex rescues CSD facilitation. Cef-treatment increased slightly, but significantly, CSD threshold without affecting CSD propagation. In the second approach I investigated whether pharmacological reduction of the glutamate clearance in WT mice to a value similar to that of FHM2-KI mice lowered the threshold for CSD induction and increased the velocity of CSD propagation. I found that the concentration of a glutamate transporters inhibitor able to slow the glutamate clearance in WT mice similarly than in FHM2-KI, also lowered the threshold for CSD induction similarly than in FHM2-KI and increased the velocity of CSD propagation, but less than in FHM2-KI, suggesting that the reduced rate of glutamate clearance can account for most of the facilitation of CSD induction and for a fraction of the facilitation of CSD propagation in the FHM2 mutants. Given the key role of N-methyl-D-aspartate receptors (NMDARs) in CSD initiation, I investigated whether in FHM2-KI mice there is an excessive NMDARs activation due to increased glutamate spillover. Measurements of post-synaptic NMDAR currents (NMDARs-EPSC) elicited in cortical layer 2/3 pyramidal neurons (Pyrs) by extracellular stimulation in layer 1 in acute cortical slices of the barrel cortex revealed an increased amplitude and a slowing of the decay of the NMDARs-EPSC in FHM2-KI mice. Quantitatively, the changes in FHM2-KI were larger after trains of pulses at high frequency than after a single stimulus. These data support the conclusion that defective glutamate clearance in FHM2 leads to increased activation of Pyrs NMDARs consequent to increased glutamate spillover, and suggest that this may largely account for CSD facilitation in FHM2. I investigated the pharmacological profile of the NMDARs activated by increased glutamate spillover. I found that in WT mice the large majority of the NMDARs-EPSC is due to activation of triheteromeric GluN2A-2BRs and only a small fraction is due to activation of diheteromeric GluN2BRs, while in FHM2-KI a larger fraction of the NMDARs-EPSC is due to activation of GluN2BRs compared to WT mice. This is consistent with preferential recruitment of GluN2BRs by increased glutamate spillover in FHM2 KI mice. The comparison of the pharmacological data suggests that the increased activation of GluN2BRs might quantitatively account for most, if not all, the increased activation of NMDARs in FHM2-KI compared to WT mice. These findings in FHM2 KI, together with previous findings in FHM1 mouse model, support the idea that excessive glutamatergic transmission and excessive activation of NMDARs play a crucial role in CSD facilitation in FHM mouse models. Previous findings obtained in the lab revealed unaltered inhibitory transmission at several cortical inhibitory synapses in contrast with increased excitatory transmission at cortical Pyrs synapses, together with the α2NKA expression at excitatory but not inhibitory cortical synapses, point to a dysfunctional regulation of E/I balance in FHM. As a first test of this hypothesis, we decided to investigate whether FHM mutations alter the dynamic regulation of the E/I balance in L2/3 during recurrent network activity induced by optogenetic activation of L2/3 Pyrs with different types of light stimuli, that presumably mimic different types of physiological activity. I performed in utero electroporation at day 15.5 of gestation allows to selectively express channelrhodopsin-2 (ChR2), a non-specific cation channel that depolarize upon blue light illumination (443 nm), in L2/3 Pyrs; this allows to selectively activate L2/3 Pyrs in thalamocortical slices and to record light-evoke excitatory and inhibitory currents in voltage-clamped neurons not expressing ChR2. My work in the last period was dedicated to implement this technique in particular, I optimized I) the concentrations of cDNA and Fast Green FCF to be injected into the embryo; II) the electroporation and injection parameters; III) the mating strategy in order to obtain a sufficient number of pregnant mice and surviving pups after electroporation. If we succeed in resolving the remaining technical problems regarding the electroporation method, we are now ready to investigate whether FHM mutations alter the dynamic regulation of the E/I balance in L2/3 during recurrent network activity. |
