Fast Ca²⁺ responses in astrocyte end‐feet and neurovascular coupling in mice

Abstract

Cerebral blood flow (CBF) is regulated by the activity of neurons and astrocytes. Understanding how these cells control activity-dependent increases in CBF is crucial to interpreting functional neuroimaging signals. The relative importance of neurons and astrocytes is debated, as are the functional implications of fast Ca changes in astrocytes versus neurons. Here, we used two-photon microscopy to assess Ca changes in neuropil, astrocyte processes, and astrocyte end-feet in response to whisker pad stimulation in mice. We also developed a pixel-based analysis to improve the detection of rapid Ca signals in the subcellular compartments of astrocytes. Fast Ca responses were observed using both chemical and genetically encoded Ca indicators in astrocyte end-feet prior to dilation of arterioles and capillaries. A low dose of the NMDA receptor antagonist (5R,10s)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine-hydrogen-maleate (MK801) attenuated fast Ca responses in the neuropil and astrocyte processes, but not in astrocyte end-feet, and the evoked CBF response was preserved. In addition, a low dose of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), an agonist for the extrasynaptic GABA receptor (GABA R), increased CBF responses and the fast Ca response in astrocyte end-feet but did not affect Ca responses in astrocyte processes and neuropil. These results suggest that fast Ca increases in the neuropil and astrocyte processes are not necessary for an evoked CBF response. In contrast, as local fast Ca responses in astrocyte end-feet are unaffected by MK801 but increase via GABA R-dependent mechanisms that also increased CBF responses, we hypothesize that the fast Ca increases in end-feet adjust CBF during synaptic activity.

Cortical Circuit Activity Evokes Rapid Astrocyte Calcium Signals on a Similar Timescale to Neurons

“…The direct comparison of astrocyte-neuron temporal dynamics was facilitated by the use of two different GECIs to properly segregate sub-cellular compartments in each cell type. This presents an advantage over previous studies using a single calcium indicator, which required data fitting and interpolation to detect fast astrocyte signals (Lind et al, 2013(Lind et al, , 2018. We also grouped neuronal ROIs into high, mid, and low responders (Margolis et al, 2012) based on their amplitudes and found that high responders were the most correlated and closely associated with active Lck-GCaMP6f ROIs (Figures S3F and S3G).…”

“…Astrocyte calcium signaling plays a pivotal role in the modulation of synapses (Araque et al, 2014) and local blood flow (Attwell et al, 2010); however, the temporal dynamics of these signals is currently debated (Ding et al, 2013;Gee et al, 2014;Lind et al, 2013Lind et al, , 2018Nizar et al, 2013;Otsu et al, 2015;Wang et al, 2006;Winship et al, 2007), calling into question the function of astrocytes in these brain processes. Many of these studies have focused on cytosolic calcium signals, primarily in astrocyte somata (Ding et al, 2013;Lind et al, 2013;Nizar et al, 2013;Wang et al, 2006;Winship et al, 2007), but recent work with genetically encoded calcium indicators (GECIs) has identified localized calcium signals within fine astrocyte processes (Agarwal et al, 2017;Bindocci et al, 2017;Lind et al, 2018;Otsu et al, 2015;Srinivasan et al, 2015;Stobart et al, 2018). These microdomains (MDs) can be driven by activation of the circuit and nearby neurons (Bindocci et al, 2017;Stobart et al, 2018) and they may be more relevant for information processing than large-scale calcium events.…”

“…These microdomains (MDs) can be driven by activation of the circuit and nearby neurons (Bindocci et al, 2017;Stobart et al, 2018) and they may be more relevant for information processing than large-scale calcium events. Fast astrocyte process calcium signals have previously been reported in the CNS of anesthetized mice (Lind et al, 2013(Lind et al, , 2018Otsu et al, 2015), but the evidence for temporal coupling of astrocyte MDs and neurons in awake animals is currently lacking. Furthermore, neuromodulatory pathways (such as the locus coeruleus and nucleus basalis) can evoke large-scale astrocyte calcium responses through mechanisms mediated by G-protein-coupled receptors (GPCRs) and inositol-1,4,5-trisphosphate (IP 3 ) type 2 receptors (IP 3 R2; Chen et al, 2012;Ding et al, 2013;Paukert et al, 2014;Srinivasan et al, 2015;Takata et al, 2011), and this can enhance the responses of astrocytes (Paukert et al, 2014) and neurons to local circuit activity (Chen et al, 2012;Takata et al, 2011).…”

Augmented astrocyte microdomain Ca2+ dynamics and parenchymal arteriole tone in angiotensin II‐infused hypertensive mice

“…Astrocyte function is compartmentalized, and little is known about the effect disease conditions have on the homeostasis and function of distinct astrocyte compartments (Agarwal et al, ; Dunn et al, ; Lind et al, ; Oheim, Schmidt, & Hirrlinger, ; Rungta et al, ; Shigetomi et al, ). Using saline and Ang II‐infused GLAST‐CreERT2; R26‐lsl‐GCaMP3 mice, where compartmentalized Ca 2+ activity can be readily assessed, we show increased spontaneous astrocyte Ca 2+ events in MD of hypertensive mice.…”

Sensory Stimulation-Induced Astrocytic Calcium Signaling in Electrically Silent Ischemic Penumbra

“…Neurons and astrocytes generally respond to somatosensory stimulation with intensely increased Ca 2+ in neuronal somas, neuropil, astrocytic somas, processes, and end-feet (Khennouf et al, 2016; Jessen et al, 2017; Lind et al, 2018; Stobart et al, 2018). Since stroke reduces spontaneous neuronal Ca 2+ activity in adult but not aged mice, (Fordsmann et al, 2018) we next assessed how neurons in the electrically silent penumbra reacted to somatosensory stimulation during the early post-stroke period.…”