Astrocytic GPCR-Induced Ca2+ Signaling Is Not Causally Related to Local Cerebral Blood Flow Changes.

Activation of Gq-type G protein-coupled receptors (GPCRs) gives rise to large cytosolic Ca2+ elevations in astrocytes. Previous in vitro and in vivo studies have indicated that astrocytic Ca2+ elevations are closely associated with diameter changes in the nearby blood vessels, which astrocytes enwrap with their endfeet. However, the causal relationship between astrocytic Ca2+ elevations and blood vessel diameter changes has been questioned, as mice with diminished astrocytic Ca2+ signaling show normal sensory hyperemia. We addressed this controversy by imaging cortical vasculature while optogenetically elevating astrocyte Ca2+ in a novel transgenic mouse line, expressing Opto-Gq-type GPCR Optoα1AR (Astro-Optoα1AR) in astrocytes. Blue light illumination on the surface of the somatosensory cortex induced Ca2+ elevations in cortical astrocytes and their endfeet in mice under anesthesia. Blood vessel diameter did not change significantly with Optoα1AR-induced Ca2+ elevations in astrocytes, while it was increased by forelimb stimulation. Next, we labeled blood plasma with red fluorescence using AAV8-P3-Alb-mScarlet in Astro-Optoα1AR mice. We were able to identify arterioles that display diameter changes in superficial areas of the somatosensory cortex through the thinned skull. Photo-stimulation of astrocytes in the cortical area did not result in noticeable changes in the arteriole diameters compared with their background strain C57BL/6. Together, compelling evidence for astrocytic Gq pathway-induced vasodiameter changes was not observed. Our results support the notion that short-term (<10 s) hyperemia is not mediated by GPCR-induced astrocytic Ca2+ signaling.

Virally induced CRISPR/Cas9-based knock-in of fluorescent albumin allows long-term visualization of cerebral circulation in infant and adult mice.

Albumin, a protein produced by liver hepatocytes, represents the most abundant protein in blood plasma. We have previously engineered a liver-targeting adeno-associated viral vector (AAV) that expresses fluorescent protein-tagged albumin to visualize blood plasma in mice. While this approach is versatile for imaging in adult mice, transgene expression vanishes when AAV is administered in neonates due to dilution of the episomal AAV genome in the rapidly growing liver. Here, we use CRISPR/Cas9 genome editing to insert the fluorescent protein mNeonGreen (mNG) gene into the albumin (Alb) locus of hepatocytes to produce fluorescently labeled albumin (Alb-mNG). We constructed a CRISPR AAV that includes ∼1 kb homologous arms around Alb exon 14 to express Alb-mNG. Subcutaneous injection of this AAV with AAV-CMV-Cas9 in postnatal day 3 mice resulted in two-photon visualization of the cerebral cortex vasculature within ten days. The expression levels of Alb-mNG were persistent for at least three months and were so robust that vasomotion and capillary blood flow could be assessed transcranially in early postnatal mice. This knock-in approach provides powerful means for micro- and macroscopic imaging of cerebral vascular dynamics in postnatal and adult mice.

Liver-secreted fluorescent blood plasma markers enable chronic imaging of the microcirculation.

Studying blood microcirculation is vital for gaining insights into vascular diseases. Blood flow imaging in deep tissue is currently achieved by acute administration of fluorescent dyes in the blood plasma. This is an invasive process, and the plasma fluorescence decreases within an hour of administration. Here, we report an approach for the longitudinal study of vasculature. Using a single intraperitoneal or intravenous administration of viral vectors, we express fluorescent secretory albumin-fusion proteins in the liver to chronically label the blood circulation in mice. This approach allows for longitudinal observation of circulation from 2 weeks to over 4 months after vector administration. We demonstrate the chronic assessment of vascular functions including functional hyperemia and vascular plasticity in micro- and mesoscopic scales. This genetic plasma labeling approach represents a versatile and cost-effective method for the chronic investigation of vasculature functions across the body in health and disease animal models.