Poster Z19 - Characterization of astrocytic Ca2+-dependent pathways of neurovascular coupling combining optogenetics and laser doppler flowmetry

Introduction: In the last decades, astrocytes have emerged as a very important mediator in the neurovascular coupling (NVC) [1-6]. Accumulated evidence has linked glutamatergic activity in neuronal synapses with increase in astrocytic Ca2+ activity, which constitutes the essential component in all astrocytic pathways of vascular control [1]. Several experimental studies have tried to quantify the contribution of two main pathways: 1) the eicosanoids, defined by the Ca2+-dependent production of arachidonic acid (AA) through the Phospholipase A2 (PLA2) enzyme [2-5]; and 2) the K+ syphoning, characterized by the Ca2+-triggered release of K+ to the perivascular space [6]. However, these past studies have produced contradicting results due to two main factors: failure to isolate astrocytic contribution to the vascular response, and 2) most studies considered each pathway to contribute additively to the resulting cerebral blood flow (CBF) response. We hypothesize that these astrocytic-mediated pathways contribute to the CBF response in a dynamic and non-linear fashion. We believe these pathways can be fully characterized by combining experimental measurements of localized CBF in response to astrocytic Ca2+ stimulation with pharmacological manipulation to block each pathway and biophysical modeling (previous year study).

Materials and

Methods: To evaluate our hypothesis, we utilized a double transgenic mouse model based on a tetracycline gene transcription system to express Chanelrodopsin2 (ChR2) solely in cortical astrocytes [7]. Hence, astrocytic Ca2+ activity is optogenetically stimulated through a craniotomy using LED light (Figure 1B, blue to open channels and amber to close them [8]). Subsequent localized CBF response was measured via Laser Doppler Flowmetry (LDF) modality (Figure 1A). We proposed that the eicosanoid pathway encompass a population of Group IIA secretory phospholipase A2 (sPLA2) alongside Group IV cytosolic PLA2 (cPLA2). We blocked the contribution of this pathway using of Group IIA inhibitor Varespladib and Group IV inhibitor MAFP. The K+ syphoning pathway was blocked using Paxilline, a non-selective BK channel antagonist. All drugs were topically applied.

Results, Conclusions, and Discussions: After blocking cPLA2 mediated pathways we found a significant reduction in CBF during stimulation suggesting an active role in vasodilation (Figure 1C). Combination of MAFP with all other drugs also produced dissimilar CBF dynamics that reflect the non-linear nature of astrocytic mediated NVC. A decrease in vasoconstriction after application of MAFP and Varespladib together compared to MAFP alone supports our hypothesis about the presence of sPLA2 population in the eicosanoid pathway. The remaining positive response found after application of all drugs might suggest the presence of another pathway that was not considered. Additional evaluation of nitric oxide (NO) release by astrocytes is ongoing. Furthermore, isolated inhibition of sPLA2 and BK channels induced oscillatory behavior in the CBF baseline following drug application implying a pathway contribution towards maintaining vascular tone (Figure 1D).

Acknowledgements (Optional): Thanks to the Wallace Coulter Foundation for funding this study