In vivo measurement of astrocytic endfoot Ca2+ and parenchymal vessel responses during 4-AP induced epilepsy using two-photon fluorescence lifetime microscopy

Abstract

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.