Shining light on the role of Parvalbumin interneurons in cortical spreading depression

As a pathological phenomenon, Cortical Spreading Depression (CSD) is the most likely cause of migraine aura. CSD has also been implicated in the physiopathology of traumatic brain injury and ischemic/hemorrhagic stroke. It is even likely that after traumatic brain injury, depolarization waves such as CSD exacerbate brain damage. Using electrophysiological recordings and DC-coupled amplification this process can be visualised as slow and large negative potential drops with amplitudes in the range of -5 to -25 mV. While the macroscopic changes related to CSD are now clear, microscopic mechanisms and cell-cell interactions need further study.Optogenetics is an efficient and well-established method in which viral vectors are used to induce expression of light-sensitive ion channels like Channelrhodopsin-2 (ChR2) into the membranes of specific cell types, such as neurons, allowing the researcher to manipulate the ion fluxes of these cells independently using light only.Our starting hypothesis was that suppressing inhibitory neurons will modulate velocity and frequency of CSD. Since 50 % of the GABA-ergic cortical interneurons express parvalbumin (PV), we focused on investigating PV(+) interneurons. In the experimental group we injected a Cre-dependent vector (AAV2/7-CMV-FLEX-ChR2-mCherry) in adult P120 PV-ires-cre mice (C57Bl/6J background, n=5) in order to get cell-specific ChR2 expression in PV(+) interneurons. We used a total of 6 C57Bl/6J mice (4 injected with a nonspecific neuronal vector AAV2/7-CMV-ChR2-eGFP and 2 blanks) as control group. All animals were injected in the left hemisphere of the visual cortex (-3.2mm AP, 2.5mm LM (BR); depth 400 um; 15x50 nl; Drumond Nanoject II) and recovered at least for 4 weeks. CSD was induced in an acute preparation as follows. Animals were anesthetized with a combination of urethane and chlorprothixene. Two craniotomies for each hemisphere (4 in total) were prepared having one positioned above the visual cortex and one above the prefrontal cortex. CSD waves were constantly induced by placing cotton balls on the visual cortices which were impregnated with 1M KCl solution. CSD waves were recorded in the prefrontal cortex using Ag/AgCl electrodes embedded in glass capillaries and an optical fiber was positioned above the visual cortex of the transduced hemisphere for laser stimulation (10 Hz, 50mW/mm², 60 mins).Our results so far demonstrate that there is no effect on the duration of CSD waves between the two mouse strains or upon an optogenetic activation of either the complete set of neurons or the PV(+) interneurons subtype. In contrast, we found an effect caused by the genetic background when comparing C57Bl/6J mice and PV-Cre mice where in the latter genotype CSD waves propagate with a higher frequency. In addition, optogenetic activation of PV(+) interneurons increases the frequency even further. These results suggest both the involvement of the genetic background and the role of the excitation-inhibition balance on CSD initiation and propagation. Implementing the optogenetics toolbox in CSD research provides a unique opportunity to look into cell type-specific mechanisms of CSD onset/propagation and can facilitate dissecting different physiopathological feedback loops.Layman’s abstract Migraine aura is usually a visual, yet sometimes sensory, motor or verbal disturbance preceding the attack. This mysterious phenomenon can be linked to cortical spreading depression (CSD), a slowly migrating wave of pathological brain activity. To study the mechanism of CSD we genetically modified specific neurons in the mouse cortex allowing their direct activation by short, focused laser pulses (optogenetics). In parallel we induced CSD waves chemically and recorded their passage with implantable electrodes. Activation of these neurons increased the frequency and variability of CSD waves. Unravelling these microscopic mechanisms is paramount to eventually develop better treatments for migraine with aura. Layman’s abstract (Dutch)De aura die vaak een migraine aanval voorafgaat, veroorzaakt visuele, maar ook sensorische, motorische, of verbale verstoringen. Het is potentieel gelinkt aan een traag migrerende golf van pathologisch verhoogde hersenschors-activiteit (cortical spreading depression, (CSD)). Om het mechanisme achter CSD te bestuderen, werden specifieke cellen in de muis-cortex genetisch gemodificeerd, waardoor ze rechtstreeks activeerbaar worden door korte, gefocuste laser pulsen (optogenetica). Tegelijkertijd induceerden we chemisch CSD golven, opgemeten met geïmplanteerde elektrodes. Optogenetische activatie van deze neuronen verhoogde de frequentie en variabiliteit van CSD golven. Kennis van deze microscopische mechanismen is cruciaal voor de ontwikkeling van betere behandelingen voor migraine met aura.Résumé en Français:La propagation de la dépression corticale (CSD) est une onde. de dépolarisation de neurones, d'origine pathologique se déplacant lentement et qui est à la base de la migraine avec aura mais est aussi impliquée lors de traumatisme crânien. Afin d’analyser de manière précise quels étaient les sous-populations de neurones responsables de cette onde de dépolarisation, nous avons utilisé sur des souris la technique dite d’optogénétique qui permet de visualiser l’activité de neurones ciblés. Nos résultats suggèrent qu’à la fois le patrimoine génétique et l’équilibre entre excitation-inhibition joue un rôle lors de l’initiation et la propoagation de la CSD.status: publishe