Differential pial and penetrating arterial responses examined by optogenetic activation of astrocytes and neurons

Abstract

A variety of brain cells participates in neurovascular coupling by transmitting and modulating vasoactive signals. The present study aimed to probe cell type-dependent cerebrovascular (i.e., pial and penetrating arterial) responses with optogenetics in the cortex of anesthetized mice. Two lines of the transgenic mice expressing a step function type of light-gated cation channel (channelrhodopsine-2; ChR2) in either cortical neurons (muscarinic acetylcholine receptors) or astrocytes (Mlc1-positive) were used in the experiments. Photo-activation of ChR2-expressing astrocytes resulted in a widespread increase in cerebral blood flow (CBF), extending to the nonstimulated periphery. In contrast, photo-activation of ChR2-expressing neurons led to a relatively localized increase in CBF. The differences in the spatial extent of the CBF responses are potentially explained by differences in the involvement of the vascular compartments. In vivo imaging of the cerebrovascular responses revealed that ChR2-expressing astrocyte activation led to the dilation of both pial and penetrating arteries, whereas ChR2-expressing neuron activation predominantly caused dilation of the penetrating arterioles. Pharmacological studies showed that cell type-specific signaling mechanisms participate in the optogenetically induced cerebrovascular responses. In conclusion, pial and penetrating arterial vasodilation were differentially evoked by ChR2-expressing astrocytes and neurons.

Keywords: Animal model; cerebral microcirculation; in vivo optical imaging; laser speckle flowgraphy; two-photon microscopy.

Figure 1.

Laser speckle flowgraphy (LSFG) measurements of CBF responses to transcranially delivered photo-activation of ChR2 in the cortex of anesthetized mice. (a) A representative raw image of photograph that captured collimated blue LED stimulation on the skull of the mouse (ChR2-astrocyte). Two circular ROIs (region of interest) were defined for the LSFG measurements at the stimulated core (0.5 mm in diameter) and periphery (1.5–2.0 mm away from the center of the stimulated spot). A map of normalized intensity of blue LED illumination (b) was reconstructed from the photograph (a). Scale bar: 1 mm. (c) A representative map of LSFG in the same animal shown in (a). A relative change in CBF from the pre-stimulation baseline represents a widespread increase in evoked CBF, while the blue LED illumination (0.26 mW/mm²) was focally delivered. (d, e) Spatial extent of light intensity of blue LED illumination (black dot) and CBF responses (black line; averaged from 30 to 40 sec after the onset of stimulation) relative to the stimulated spot were compared in ChR2-astrocyte (d) and ChR2-neuron (e) mice. Dark gray areas represent the locations of two ROIs: the core (C) and periphery (P). The spot size of the blue LED illumination measured at full-width at half maximum (arrows) was 0.5 mm. The temporal dynamics of average changes in evoked CBF measured at the core (black) and periphery (gray) were compared. Photo-activation with 3 sec of blue LED illumination followed by 3 sec of yellow LED illumination was applied at time 0.

Figure 2.

Comparisons of response amplitude of CBF responses and their spatial extent with changes in the power of photo-activation. (a) Representative results of LSFG measurements with varying intensities of blue LED illumination. Upper panels show the results of the representative ChR2-astrocyte mouse (ChR2-A), and lower panels show results from the ChR2-neuron mouse (ChR2-N). The locations of photo-activated regions are depicted on the pre-stimulation baseline images of LSFG (white dots, left panel). The color scale shows a mean blur rate (MBR). Photo-activation with 0.005 mW/mm² did not result in detectable changes in CBF. The evoked CBF responses and their spatial extent were monotonically increased as the intensity increased over the range of 0.025–2.5 mW/mm². Scale bar: 1 mm. (b) Comparison of spatial profiles of CBF responses between ChR2-astrocyte (ChR2-A, upper) and ChR2-neuron (ChR2-N, lower) mice. Individual lines indicate the average responses of evoked CBF over all animals, and the color indicates different power of photo-stimulation. With an increase in photo-stimulation, a higher peak amplitude was observed for ChR2-neuron mice, whereas a broader response was evident in ChR2-astrocyte mice. (c) Quantitative comparisons of the response amplitude measured at the stimulated core. The response amplitude nonlinearly increased as the power of photo-activation increased. Photo-activation of ChR2-neurons (gray triangle, N = 5 animals) showed consistently larger responses (p < 0.05) relative to ChR2-astrocyte activation (black circle, N = 5 animals). A regression line: Y = 0.13 LN (X) + 0.86 and Y = 0.08 LN (X) + 0.86 for ChR2-neuron and ChR2-astrocyte, respectively. (d) Comparisons of the spatial extent of the CBF responses. For all stimulation power, equivalent spatial extent was observed between ChR2-astrocyte (black circle) and ChR2-neuron (gray triangle) mice. A regression line: Y = 0.08 LN (X) + 0.91 and Y = 0.09 LN (X) + 0.85 for ChR2-neuron and ChR2-astrocyte, respectively. (e) Relationship between the peak amplitude of CBF responses and their spatial extents. Significantly higher spatial extent (p < 0.05) normalized by the peak amplitude was consistently observed for ChR2-astrocyte activation (black circle) compared to ChR2-neuron activation (gray triangle). A plot indicates the mean response to each power of photo-stimulation in individual animals. A regression line: Y = 0.81 X + 0.03 and Y = 1.4 X – 0.4 for ChR2-neuron (R = 0.47, p < 0.05) and ChR2-astrocyte mice (R = 0.62, p < 0.05), respectively.

Figure 3.

Changes in the diameter of cerebral arteries in response to the photo-activation of ChR2-astrocytes. (a) Representative two-photon microscopy images of YFP-expressing ChR2 (green) and sulforhodamine-101 (SR101) stained blood vessels (red) in the ChR2-astrocyte mouse. XYZ serial images were captured at depths of 0–400 µm with intervals of 100 µm from the cortical surface. A surface pial artery (SA) and terminal arteriole (TA) that enter the cortex (white rectangle) were identified at a depth of 0 µm (top panels), and the same penetrating arteriole (PA) was traced deeper in the cortex (white rectangle). Pre-stimulation baseline and post-stimulation images of the microvasculature were compared (center and right panels, respectively). All arteries (SA, TA, and PA) showed remarkable vasodilation (arrowheads) after photo-stimulation, whereas other microvessels remained unchanged. (b) Representative temporal dynamic responses of the arterial diameters shown in (a). The images were captured every 5 sec, and photo-activation was applied for 3 sec with an instrumental mercury lamp (470–490 nm, an average power of 0.26 mW/mm²) to the field of view at a depth of 0 µm without scanning to protect the photodetectors. Both the SA (black) and TA (gray) showed similar dynamic changes (left panel), which were also preserved in the responses of PA-1, 2, 3, and 4 measured at depths of 100, 200, 300, and 400 µm, respectively (dark to light gray, right panel). (c) Comparison of the changes in the diameter in response to the photo-activation of ChR2-astrocytes (N = 5 animals). Statistically significant differences in the vasodilation of the penetrating arterioles and the pial arteries were not observed (p > 0.05; Dunnett’s test). Each symbols represent the same animal results in this animal group. (d) Comparison of YFP-expressing ChR2 densities at various cortical depths. The area density of the YFP signals (ChR2) was calculated in each image using a k-means classification method. The area density of the ChR2-positve astrocytes showed statistically significant differences (*p < 0.05; Dunnett’s test) in the cortical surface relative to the parenchyma measured at depths of 100–400 µm (N = 3 animals).

Figure 4.

Changes in the diameters of cerebral arteries in response to the photo-activation of ChR2-neurons. (a) Representative two-photon microscopy images of YFP-expressing ChR2 (green) and sulforhodamine-101 (SR101) stained blood vessels (red) in the ChR2-neuron mouse. As shown in Figure 3, a surface pial artery (SA) and terminal arteriole (TA) that enter the cortex (white rectangle) were identified at a depth of 0 µm (top panels), and the same penetrating arteriole (PA) was traced deeper in the cortex (white rectangle). Remarkable vasodilation of the PA was observed after photo-stimulation (arrowheads). (b) Representative dynamic temporal responses of the arterial diameters shown in (a). The images were captured every 5 sec, and photo-activation was applied with 3 sec of illumination with an instrumental mercury lamp (470–490 nm, an average power of 0.26 mW/mm²). In contrast to the ChR2-astrocyte activation, changes in the diameters of the SA (black) were small, while the TA (gray) showed substantial vasodilation (left panel). Additionally, temporal dynamics of the changes in the diameters of PAs varied, depending on the depths at which they were measured; PA-1, 2, 3, and 4 were measured at depths of 100, 200, 300, and 400 µm, respectively (dark to light gray, right panel). (c) Comparison of the changes in the diameters in response to photo-activation of ChR2-neurons (N = 6 animals). Statistically significant differences in the vasodilatory responses of PA were detected compared to the changes in the diameters of SA (*p < 0.05; Dunnett’s test). Each symbols represent the same animal results in this animal group. (d) Comparison of YFP-expressing ChR2 densities at various cortical depths. Statistically significant differences (*p < 0.05; Dunnett’s test) in the YFP-expressing neural ChR2 densities were detected at depths of 100 µm and 200 µm compared to the surface (N = 3 animals).

Figure 5.

Comparison of the pial and penetrating arterial vasodilation. (a) Pre-stimulation baseline diameters of pial (Pia) and penetrating (Pen) arteries (averaged over depths of 100–400 µm) measured in ChR2-astrocyte (As) and ChR2-neuron (Ne) mice. Each symbols represent the same animal results in the animal groups shown in Figures 3(c) and 4(c). (b) Relative changes in diameters induced by photo-stimulation. (c) A ratio of the diameter responses of pial and penetrating arteries (i.e., relative changes in diameter of the penetrating arterioles divided by those of the pial arteries). Significantly higher dilation in the penetrating arterioles relative to the pial arteries were observed for ChR2-neuron (ChR2-N) mice compared to ChR2-astrocyte (ChR2-A) mice (*p < 0.05).

Figure 6.

Pharmacological inhibition of the CBF responses to photo-activation support the findings that different vasoactive pathways were involved in a cell type-specific manner. CBF responses to the photo-activation of ChR2-astrocytes (ChR2-A, dark gray) were significantly suppressed (*p < 0.05, paired t-test, pre- vs. post-treatment) by topical application of MPEP, carbenoxolone (CBX), barium chloride (BaCl₂), and MK801, although these drugs did not exert significant effects (p > 0.05) on the responses to ChR2-neuron activation (ChR2-N, light gray). The CBF responses to the photo-activation of ChR2-neurons were significantly suppressed by atropine, tetrodotoxin (TTX), indomethacin (Ind), L-NAME, and paxilline (Pax), whereas significant effects on the CBF responses to ChR2-astrocyte activation were not detected after treatments with these drugs. Error bar: standard deviation.