Abstract
Astrocytic Ca2+ fluctuations associated with functional hyperemia have typically been measured from large cellular compartments such as the soma, the whole arbor and the endfoot. The most prominent Ca2+ event is a large magnitude, delayed signal that follows vasodilation. However, previous work has provided little information about the spatio-temporal properties of such Ca2+ transients or their heterogeneity. Here, using an awake, in vivo two-photon fluorescence-imaging model, we performed detailed profiling of delayed astrocytic Ca2+ signals across astrocytes or within individual astrocyte compartments using small regions of interest next to penetrating arterioles and capillaries along with vasomotor responses to vibrissae stimulation. We demonstrated that while a 5-s air puff that stimulates all whiskers predominantly generated reproducible functional hyperemia in the presence or absence of astrocytic Ca2+ changes, whisker stimulation inconsistently produced astrocytic Ca2+ responses. More importantly, these Ca2+ responses were heterogeneous among subcellular structures of the astrocyte and across different astrocytes that resided within the same field of view. Furthermore, we found that whisker stimulation induced discrete Ca2+ “hot spots” that spread regionally within the endfoot. These data reveal that astrocytic Ca2+ dynamics associated with the microvasculature are more complex than previously thought, and highlight the importance of considering the heterogeneity of astrocytic Ca2+ activity to fully understanding neurovascular coupling.
Highlights
Functional hyperemia is a fundamental control mechanism that provides a rapid local increase in blood flow in response to increased neuronal activity
We extended these analyses, examining Ca2+ dynamics in as many astrocytes within the field of view as possible and monitoring endfoot Ca2+ changes associated with penetrating arterioles and capillaries in the same cortical layer and focal plane
Ca2+ rises were observed in the presence of vasodilation; (3) sensory stimulation did not elicit a global rise in endfoot Ca2+, but instead triggered discrete Ca2+ “hot spots” that typically spread around the endfoot and occasionally remained localized
Summary
Functional hyperemia is a fundamental control mechanism that provides a rapid local increase in blood flow in response to increased neuronal activity. Our previous study has reported that the delayed astrocytic endfoot Ca2+ signal is mediated by both neurons and vasculature, suggesting a complex interplay between multiple mechanisms that must temporally and spatially coincide to cause a large activation of endfeet This intriguing finding necessitates further detailed analysis of endfoot Ca2+ dynamics to gain insights into their contributions to functional hyperemia (Tran et al, 2018). Our own work in awake and active animals using astrocyte Rhod-2 AM, GCaMP3, or GCaMP6s, revealed that whisker stimulation elicited large astrocytic Ca2+ signals that followed rather than preceded vasodilation (Tran et al, 2018) These astrocytic endfoot Ca2+ events were mediated by both glutamatergic transmission and vascular-derived nitric oxide. We characterized the cortical astrocyte Ca2+ dynamics, in particular astrocytic endfoot Ca2+, and examined its relationship with functional hyperemia in completely awake mice in vivo using two-photon imaging
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