In Vivo measurement of astrocytic endfoot Ca~(2+) and parenchymal vessel responses during 4-AP induced epilepsy using two-photon lifetime microscopy

摘要

Neurovascular coupling (NVC) is defined as a local increase in cerebral blood flow in response to neuronal activity, it forms the basis of functional brain imaging and is altered during epilepsy. Because astrocytic calcium signaling (Ca~(2+)) has been involved in the response of parenchymal vessels, this study investigates the role of this pathway during epilepsy. We exploit 4-Aminopyridine (4-AP) induced epileptic seizures to show that absolute Ca~(2+) concentration in astrocytic endfeet correlates with the changes in diameter of parenchymal vessels during neural activity in vivo. A two-photon laser scanning fluorescence lifetime microscopy was developed to simultaneously monitor free Ca~(2+) concentration in astrocytic endfeet with the calcium-sensitive indicator Oregon Green 488 BAPTA-1 (OGB-1) and the diameter of parenchymal vessels in the somatosensory cortex of mice following 4-AP injection. Our results reveal that the resting Ca~(2+) concentration in glial cells was spatially heterogeneous and that resting Ca~(2+) concentration in somatic regions was significantly higher than in endfoot regions. Moreover, following 4-AP injection in the somatosensory cortex of mice, we observed increases of Ca~(2+) in astrocytic endfeet associated with vasodilation of parenchymal vessels for each individual ictal event in the epileptic focus. However, vasodilation was seen to be inhibited by increase in absolute resting Ca~(2+) concentration. Our results suggest a role for baseline astrocytic Ca~(2+) concentration in vasodilation.